Novel proteins specific for angiogenesis

ABSTRACT

The present disclosure provides hNGAL muteins that bind Ang-2 and can be used in various application including pharmaceutical applications, for example, to inhibit or reduce angiogenesis. The present disclosure also concerns methods of making one or more muteins described herein as well as compositions and combinations comprising one or more of such muteins. The present disclosure further relates to nucleic acid molecules encoding such muteins and to methods for generation of such muteins and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of these muteins as well as compositions and combinations comprising one or more of such muteins.

I. BACKGROUND

Angiogenesis, the formation of new blood vessels from existing ones, isessential to many physiological and pathological processes. Normally,angiogenesis is tightly regulated by pro- and anti-angiogenic factors,but in the case of diseases such as cancer, ocular neovascular diseases,arthritis, and psoriasis, the process can go awry. Folkman, J., Nat.Med., 1:27-31 (1995). There are a number of diseases known to beassociated with deregulated or undesired angiogenesis. Such diseasesinclude, but are not limited to, ocular neovascularisation, such asretinopathles (including diabetic retinopathy), age-related maculardegeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis,inflammatory disease, such as a rheumatoid or rheumatic inflammatorydisease, especially arthritis (including rheumatoid arthritis), or otherchronic inflammatory disorders, such as chronic asthma, arterial orpost-transplantational atherosclerosis, endometriosis, and neoplasticdiseases, for example so-called solid tumors and liquid (orhematopoletic) tumors (such as leukemias and lymphomas). Other diseasesassociated with undesired angiogenesis will be apparent to those skilledin the art.

Although many signal transduction systems have been implicated in theregulation of angiogenesis, one of the best-characterized and mostendothelial cell-selective systems involves the Tie-2 receptor tyrosinekinase that is selectively expressed within the vascular endothelium(referred to as “Tie-2” or “Tie-2R” (also referred to as “ORK”); murineTie-2 is also referred to as “tek”) and its ligands, the angiopoietins(Yancopoulos, G. D., et al., Nature 407 [2000] 242-48; Gale, N. W. andYancopoulos, G. D., Genes Dev. 13:1055-1066 [1999]).

There are 4 known angiopoietins; angiopoietin-1 (“Ang-1,” alternativelyabbreviated with ANGPT1 or Ang1) through angiopoietin-4 (“Ang-4”). Theseangiopoietins are also referred to as “Tie-2 ligands” (Davis, S., etal., Cell, § 7:1161-1169 [1996]; Grosios, K., et al, Cytogenet CellGenet, § 4:118-120 [1999]; Holash, J., et al, InvestigativeOphthalmology & Visual Science, 42:1611-1625 [1999]; Koblizek, T. I., etal, Current Biology, S:529-532 [1998]; Un, P., et al, Proc Natl Acad SciUSA, 95:8829-8834 [1998]; Maisonplerre, P. C, et al, Science, 277:55-60[1997]; Papapetropoulos, A., et al, Lab Invest, 79:213-223 [1999]; Sato,T. N., et al, Nature, 375:70-74 [1998]; Shyu, K. G., et al, Circulation,95:2081-2087 [1998]; Suri, C. et al, Cell, <37:1171-1180 [1996]; Suri,C, et al, Science, 252:468-471 [1998]; Valenzuela, D. M., et al,Proceedings of the National Academy of Sciences of the USA, 96:1904-1909[1999]; Witzenbichler, B., et al, J Biol Chem, 273:18514-18521 [1998]).

Both Ang-1 and -2 bind to Tie-2 with an affinity of 3 nM (Kd)(Maisonpierre, P. C., et al., Science 277 (1997) 55-60). Whereas Ang-1binding to Tie-2 stimulates receptor phosphorylation in culturedendothelial cells, Ang-2 has been observed to both agonize andantagonize Tie-2 receptor phosphorylation (Davis, S., et al, [1996],supra; Maisonpierre, P. C., et al, [1997], supra; Kim, I, J. H. Kim, etal, Oncogene 19(39): 4549-4552 (2000); Teichert-Kuliszewska, K., P. C.Maisonpierre, et al, Cardiovascular Research 49(3): 659-70 (2001)). Thephenotypes of mouse Tie-2 and Ang-1 knockouts are similar and suggestthat Ang-1-stimulated Tie-2 phosphorylation mediates remodeling andstabilization of developing vessels in utero through maintenance ofendothelial cell-support cell adhesion (Dumont, D. J., et al, Genes &Development, 8:1897-1909 [1994]; Sato, T. N., et al, Nature, 376:10-14[1995]; Suri, C, et al, [1996], supra). The role of Ang-1 in vesselstabilization is thought to be conserved in the adult, where it isexpressed widely and constitutively (Hanahan, D., Science, 277:48-50[1997]; Zagzag, D., et al, Experimental Neurology, 59:391-400 [1999]).In contrast, Ang-2 expression is primarily limited to sites of vascularremodeling, where it is thought to block Ang-1 function, therebyinducing a state of vascular plasticity conducive to angiogenesis(Hanahan, D., [1997], supra; Holash, J., et al, Science, 284:1994-1998[1999]; Maisonpierre, P. C, et al, [1997], supra).

Human angiopoietin-2 (Ang-2) (alternatively abbreviated with ANGPT2 orAng2) is described in Maisonpierre, P. C., et al., Science 277 (1997)55-60 and Cheung. A. H., et al, Genomics 48 (1998) 389-91. Numerouspublished studies have purportedly demonstrated vessel-selective Ang-2expression in disease states associated with deregulated angiogenesis(Bunone, G., et al, American Journal of Pathology, 155:1961-1916 [1999];Etoh, T., et al, Cancer Research, 67:2145-2153 [2001]; Hangai, M., etal, Investigative Ophthalmology & Visual Science, 42:1611-1625 [2001];Holash, J., et al, [1999] supra; Kuroda, K., et al, Journal ofInvestigative Dermatology, 116:113-120 [2001]; Otani, A., et al,Investigative Ophthalmology & Visual Science, 40:1912-1920 [1999];Stratmann, A., et al, American Journal of Pathology, 153: 1459-1466[1998]; Tanaka, S., et al, J Clin Invest, 203:34-345 [1999]; Yoshida,Y., et al, International Journal of Oncology, 25:1221-1225 [1999]; Yuan,K., et al, Journal of Periodontal Research, 35:165-171 [2000]; Zagzag,D., et al, [1999] supra). An effective anti-Ang-2 therapy will benefit avast population of patients with angiogenesis-associated diseases, suchas cancer, retinopathies, arthritis, and psoriasis.

There is a great need, therefore, to identify new compounds thatspecifically recognize and bind Ang-2. Such compounds would be usefulfor diagnostic screening and therapeutic intervention in disease statesthat are associated with Ang-2 activity. Accordingly, it is an object ofthe present disclosure to provide specific binding compounds of Ang-2for modulating Ang-2 activity. Such compounds disclosed herein take theform of muteins derived from human lipocalin 2 (also known as neutrophilgelatinase associated lipocalin, “hNGAL”).

II. DEFINITIONS

The following list defines terms, phrases, and abbreviations usedthroughout the instant specification. All terms listed and definedherein are intended to encompass all grammatical forms.

As used herein, “Ang-1”, unless specified as being from a non-humanspecies (e.g., “mouse Ang-1,” “monkey Ang-1,” etc.), means human Ang-1,a full-length protein defined by Swiss Prot 015389 or a biologicallyactive fragment thereof (e.g., a fragment of the Ang-1 protein which iscapable of inducing angiogenesis in vitro or in vivo).

As used herein, “Ang-2”, unless specified as being from a non-humanspecies (e.g., “mouse Ang-2,” “monkey Ang-2,” etc.), means human Ang-2,a full-length protein defined by Swiss Prot 015123, (also see, FIG. 6 ofU.S. Pat. No. 6,166,185; incorporated herein by reference in itsentirety) or a biologically active fragment thereof (e.g., a fragment ofthe Ang-2 protein which is capable of inducing angiogenesis in vitro orin vivo).

The term “Tie-2” (also referred to in the art as “tek”) unless specifiedas being from a non-human species (e.g., “mouse Tie-2,” “monkey Tie-2,”etc.), refers to human Tie-2 or a biologically active fragment thereof.Human Tie-2 has the amino acid sequence as set forth in the NCBI proteinsequence database under Accession No. AAA61130.

As used herein, “detectable affinity” means the ability to bind to aselected target with an affinity constant of generally at least about10⁻⁵ M or below. Lower affinities are generally no longer measurablewith common methods such as ELISA and therefore of secondary importance.

As used herein, “binding affinity” of a protein of the disclosure (e.g.a mutein of human lipocalin 2) to a selected target (in the presentcase, Ang-1 or Ang-2), can be measured (and thereby KD values of amutein-ligand complex be determined) by a multitude of methods known tothose skilled in the art. Such methods include, but are not limited to,fluorescence titration, direct ELISA, competition ELISA, calorimetricmethods, such as isothermal titration calorimetry (ITC), and surfaceplasmon resonance (BIAcore). Such methods are well established in theart and examples thereof are also detailed below.

It is also noted that the complex formation between the respectivebinder and its ligand is influenced by many different factors such asthe concentrations of the respective binding partners, the presence ofcompetitors, pH and the ionic strength of the buffer system used, andthe experimental method used for determination of the dissociationconstant K_(D) (for example fluorescence titration, direct ELISA,competition ELISA or surface plasmon resonance, just to name a few) oreven the mathematical algorithm which is used for evaluation of theexperimental data.

Therefore, it is also clear to the skilled person that the K_(D) values(dissociation constant of the complex formed between the respectivebinder and its target/ligand) may vary within a certain experimentalrange, depending on the method and experimental setup that is used fordetermining the affinity of a particular mutein for a given ligand. Thismeans that there may be a slight deviation in the measured KD values ora tolerance range depending, for example, on whether the K_(D) value wasdetermined by surface plasmon resonance (Biacore), by competition ELISA,or by “direct ELISA.”

As used herein, a compound such as a mutein of the disclosure“specifically binds” a target (for example, Ang-1 or Ang-2) or has“binding specificity” for a target if it is able to discriminate betweenthat target and one or more reference targets, since binding specificityis not an absolute, but a relative property. “Specific binding” can bedetermined, for example, in accordance with Western blots, ELISA-, RIA-,ECL-, IRMA-tests, IHC and peptide scans.

The term “human lipocalin 2” or “human Lcn 2” or “human NGAL” or “hNGAL”as used herein refers to the mature human neutrophilgelatinase-associated lipocalin (NGAL) with the SWISS-PROT/UniProt DataBank Accession Number P80188. A human lipocalin 2 mutein of thedisclosure may also be designated herein as “an hNGAL mutein”. The aminoacid sequence shown in SWISS-PROT/UniProt Data Bank Accession NumberP80188 may be used as a preferred “reference sequence”, more preferablythe amino acid sequence shown in SEQ ID NO: 16 is used as referencesequence.

As used herein, a “mutein,” a “mutated” entity (whether protein ornucleic acid), or “mutant” refers to the exchange, deletion, orinsertion of one or more nucleotides or amino acids, compared to thenaturally occurring (wild-type) nucleic acid or protein “reference”scaffold. The term “mutein,” as used herein, also includes itsfunctional fragments or variants. Fragments or variants of particularmuteins described in the present disclosure preferably retain thefunction of binding to Ang-1 or Ang-2, e.g. with detectable or evenhigher affinity, and such fragments or variants are “functionalfragments or variants” of the reference mutains disclosed herein.

The term “fragment” as used herein in connection with the muteins of thedisclosure relates to proteins or peptides derived from full-lengthmature human lipocalin 2 that are N-terminally and/or C-terminallyshortened, i.e. lacking at least one of the N-terminal and/or C-terminalamino acids. Such fragments may include at least 10, more such as 20 or30 or more consecutive amino acids of the primary sequence of the maturehuman lipocalin 2 and are usually detectable in an immunoassay of themature human lipocalin 2.

In general, the term “fragment”, as used herein with respect to thecorresponding protein ligand of a mutein of the disclosure or of thecombination according to the disclosure, relates to N-terminally and/orC-terminally shortened protein or peptide ligands, which retain thecapability of the full length ligand to be recognized and/or bound by amutein according to the disclosure.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of the mature human lipocalin 2 can besubstituted by at least one amino acid that is not present at thisspecific position in the respective natural polypeptide sequence. Theterm “mutagenesis” also includes the (additional) modification of thelength of sequence segments by deletion or insertion of one or moreamino acids. Thus, it is within the scope of the disclosure that, forexample, one amino acid at a chosen sequence position is replaced by astretch of three random mutations, leading to an insertion of two aminoacid residues compared to the length of the respective segment of thewild type protein. Such an insertion or deletion may be introducedindependently from each other in any of the peptide segments that can besubjected to mutagenesis in the disclosure.

The term “random mutagenesis” means that no predetermined single aminoacid (mutation) is present at a certain sequence position but that atleast two amino acids can be incorporated with a certain probability ata predefined sequence position during mutagenesis.

“Identity” is a property of sequences that measures their similarity orrelationship. The term “sequence identity” or “identity” as used in thepresent disclosure means the percentage of pair-wise identicalresidues—following (homologous) alignment of a sequence of a polypeptideof the disclosure with a sequence in question—with respect to the numberof residues in the longer of these two sequences. Sequence identity ismeasured by dividing the number of identical amino acid residues by thetotal number of residues and multiplying the product by 100.

The term “homology” is used herein in its usual meaning and includesidentical amino acids as well as amino acids which are regarded to beconservative substitutions (for example, exchange of a glutamate residueby an aspartate residue) at equivalent positions in the linear aminoacid sequence of a polypeptide of the disclosure (e.g., any mutein ofthe disclosure).

The percentage of sequence homology or sequence identity can, forexample, be determined herein using the program BLASTP, version blastp2.2.5 (Nov. 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res.25, 3389-3402). In this embodiment the percentage of homology is basedon the alignment of the entire polypeptide sequences (matrix: BLOSUM 62;gap costs: 11.1; cutoff value set to 10⁻³) including the propeptidesequences, preferably using the wild type protein scaffold as referencein a pairwise comparison. It is calculated as the percentage of numbersof “positives” (homologous amino acids) indicated as result in theBLASTP program output divided by the total number of amino acidsselected by the program for the alignment.

Specifically, in order to determine whether an amino acid residue of theamino acid sequence of a mutein different from the wild-type humanlipocalin 2 corresponds to a certain position in the amino acid sequenceof the wild-type human lipocalin 2, a skilled artisan can use means andmethods well-known in the art, e.g., alignments, either manually or byusing computer programs such as BLAST2.0, which stands for Basic LocalAlignment Search Tool or ClustalW or any other suitable program which issuitable to generate sequence alignments. Accordingly, the wild-typehuman lipocalin 2 can serve as “subject sequence” or “referencesequence”, while the amino acid sequence of a mutein different from thewild-type human lipocalin 2 described herein serves as “query sequence”.The terms “reference sequence” and “wild type sequence” are usedinterchangeably herein.

“Gaps” are spaces in an alignment that are the result of additions ordeletions of amino acids. Thus, two copies of exactly the same sequencehave 100% identity, but sequences that are less highly conserved, andhave deletions, additions, or replacements, may have a lower degree ofsequence identity. Those skilled in the art will recognize that severalcomputer programs are available for determining sequence identity usingstandard parameters, for example Blast (Altschul, et al. (1997) NucleicAcids Res. 25, 3389-3402), Blast2 (Altschul, et al. (1990) J. Mol. Biol.215, 403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol.147, 195-197).

The term “variant” as used in the present disclosure relates toderivatives of a protein or peptide that include modifications of theamino acid sequence, for example by substitution, deletion, insertion orchemical modification. Such modifications do in some embodiments notreduce the functionality of the protein or peptide. Such variantsinclude proteins, wherein one or more amino acids have been replaced bytheir respective D-stereoisomers or by amino acids other than thenaturally occurring 20 amino acids, such as, for example, omithine,hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline.However, such substitutions may also be conservative, i.e. an amino acidresidue is replaced with a chemically similar amino acid residue.Examples of conservative substitutions are the replacements among themembers of the following groups: 1) alanine, serine, and threonine; 2)aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and6) phenylalanine, tyrosine, and tryptophan.

By a “native sequence” human lipocalin 2 is meant human lipocalin 2 thathas the same amino acid sequence as the corresponding polypeptidederived from nature. Thus, a native sequence human lipocalin 2 can havethe amino acid sequence of the respective naturally-occurring humanlipocalin 2. Such native sequence polypeptide can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence” polypeptide specifically encompassesnaturally-occurring truncated or secreted forms of the human lipocalin2, naturally-occurring variant forms such as alternatively spliced formsand naturally-occurring allelic variants of human lipocalin 2. Apolypeptide “variant” means a biologically active polypeptide having atleast about 50%, 60%, 70%, 80% or at least about 85% amino acid sequenceidentity with the native sequence polypeptide. Such variants include,for instance, polypeptides in which one or more amino acid residues areadded or deleted at the N- or C-terminus of the polypeptide. Generally avariant has at least about 70%, including at least about 80%, such as atleast about 85% amino acid sequence identity, including at least about90% amino acid sequence identity or at least about 95% amino acidsequence identity with the native sequence polypeptide.

The term “position” when used in accordance with the disclosure meansthe position of either an amino acid within an amino acid sequencedepicted herein or the position of a nucleotide within a nucleic acidsequence depicted herein. To understand the term “correspond” or“corresponding” as used herein in the context of the amino acid sequencepositions of one or more muteins, a corresponding position is not onlydetermined by the number of the preceding nucleotides/amino acids.Accordingly, the position of a given amino acid in accordance with thedisclosure which may be substituted may vary due to deletion or additionof amino acids elsewhere in a (mutant or wild-type) human lipocalin 2.Similarly, the position of a given nucleotide in accordance with thepresent disclosure which may be substituted may vary due to deletions oradditional nucleotides elsewhere in a mutein or wild type humanlipocalin 2 5′-untranslated region (UTR) including the promoter and/orany other regulatory sequences or gene (including exons and introns).

Thus, for a corresponding position in accordance with the disclosure, itis preferably to be understood that the positions of nucleotides/aminoacids may differ in the indicated number than similar neighbouringnucleotides/amino acids, but said neighbouring nucleotides/amino acids,which may be exchanged, deleted, or added, are also comprised by the oneor more corresponding positions.

In addition, for a corresponding position in a mutein based on areference scaffold in accordance with the disclosure, it is preferablyto be understood that the positions of nucleotides/amino acids arestructurally corresponding to the positions elsewhere in a mutein orwild-type human lipocalin 2, even if they may differ in the indicatednumber.

The term “organic molecule” or “small organic molecule” as used hereinfor the non-natural target denotes an organic molecule comprising atleast two carbon atoms, but preferably not more than 7 or 12 rotatablecarbon bonds, having a molecular weight in the range between 100 and2000 Dalton, preferably between 100 and 1000 Dalton, and optionallyincluding one or two metal atoms.

The word “detect”, “detection”, “detectable” or “detecting” as usedherein is understood both on a quantitative and a qualitative level, aswell as a combination thereof. It thus includes quantitative,semi-quantitative and qualitative measurements of a molecule ofinterest.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. The term “mammal” is used herein to refer to any animalclassified as a mammal, including, without limitation, humans, domesticand farm animals, and zoo, sports, or pet animals, such as sheep, dogs,horses, cats, cows, rats, pigs, apes such as cynomolgous monkeys andetc., to name only a few illustrative examples. Preferably, the mammalherein is human.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations.

A “sample” is defined as a biological sample taken from any subject.Biological samples include, but are not limited to, blood, serum, urine,feces, semen, or tissue.

The term “metastasis” according to the disclosure refers to thetransmission of cancerous cells from the primary tumor to one or moresites elsewhere in a patient where secondary tumors develop. Means todetermine if a cancer has metastasized are known in the art and includebone scan, chest X-ray, CAT scan, MRI scan, and tumor marker tests. Theterm “prevention of metastasis” means that the metastasis of theprimary, tumor or cancer is prevented, delayed, or reduced and thus thedevelopment of secondary tumors is is prevented, delayed, or reduced.Preferably the metastasis i.e secondary tumors of the lung are preventedor reduced, which means that metastatic transmission of cancerous cellsfrom the primary tumor to the lung is prevented or reduced.

The term “cancer” as used herein refers to proliferative diseases, suchas lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, billary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

The term “vascular diseases” Includes Cancer, Inflammatory diseases,Atherosclerosis, Ischemia, Trauma, Sepsis, COPD, Asthma, Diabetes, AMD,Retinopathy, Stroke, Adipositas, Acute lung injury, Hemorrhage. Vascularleak e.g. Cytokine induced, Allergy, Graves' Disease, Hashimoto'sAutoimmune Thyroiditis, Idiopathic Thrombocytopenic Purpura, Giant CellArteritis, Rheumatoid Arthritis, Systemic Lupus Erythematosus (SLE),Lupus Nephritis, Crohn's Disease, Multiple Sclerosis, UlcerativeColitis, especially to solid tumors, intraocular neovascular syndromes(such as proliferative retinopathies or age-related macular degeneration(AMD)), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J.Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev.Physiol. 53 (1991) 217-239; and Gamer, A., Vascular diseases. In:Pathobiology of ocular disease, A dynamic approach, Garner, A., andKlintworth, G. K. (eds.), 2nd edition, Marcel Dekker, New York (1994),pp 1625-1710).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains interconnected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(t)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V₁ is composedof three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the disclosure, the FRs of theanti-Ang-2 antibody (or antigen-binding portion thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)₂ fragments; (ill) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR)). Other engineered molecules,such as diabodies, triabodies, tetrabodies and minibodies, are alsoencompassed within the expression “antigen-binding fragment,” as usedherein. An antigen-binding fragment of an antibody will typicallycomprise at least one variable domain. The variable domain may be of anysize or amino acid composition and will generally comprise at least oneCDR which is adjacent to or in frame with one or more frameworksequences. In antigen-binding fragments having a V_(H) domain associatedwith a V_(L) domain, the V_(H) and V_(L) domains may be situatedrelative to one another in any suitable arrangement. For example, thevariable region may be dimeric and contain V_(H)—V_(H), V_(H)—V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

III. DESCRIPTIONS OF FIGURES

FIG. 1: demonstrates that lipocalin muteins (SEQ ID NOs: 1, 3, 7, 8, 9,and 11) are capable of blocking the interaction between human Ang-2 andits receptor Tie-2 with an IC50 in a subnanomlar range. Human Ang-2 waspre-incubated with variable concentrations of lipocalin muteins andnon-neutralized human Ang-2 was quantified on an ELISA plate withimmobilized soluble human Tie-2Fc. Benchmark antibody (SEQ ID NOs:19/20) was used as positive control. The negative control (SEQ ID NO:16) has no competitive effect. The data were fitted with a single-sitebinding model.

FIG. 2: shows the crossreactivity profile and specificity of lipocalinmuteins (SEQ ID NOs: 7 and 8) as measured in a competition ELISA format.The lipocalin muteins are crossreactive with human Ang-1, human Ang-2and mouse Ang-2. Data were fitted with a single-site binding model.

FIG. 3: demonstrates that lipocalin muteins (SEQ ID NOs: 1, 3, 6-14) arecapable of blocking the interaction between human Ang-2 and its receptorhuman Tie-2—over expressed on HEK cell. Human Ang-2 was pre-incubatedwith variable concentrations of SEQ ID NOs: 1, 3, 6-14 non-neutralizedang-2 was detected via an anti-HIS-tag antibody. Benchmark antibody (SEQID NOs: 19/20) was used as positive control. The data were fitted with asingle-site binding model.

FIG. 4: demonstrates that lipocalin muteins (SEQ ID NOs: 1, 3, 6-14) arecapable of blocking the interaction between mouse Ang-2 and the humanreceptor Tie-2 over expressed on HEK cell. Mouse Ang-2 was pre-incubatedwith variable concentrations of SEQ ID NOs: 1, 3, 6-14 andnon-neutralized mouse Ang-2 was detected via an anti-HIS-tag antibody.Benchmark antibody (SEQ ID NOs: 19/20) was used as positive control. Thedata were fitted with a single-site binding model.

FIG. 5: demonstrates that the lipocalin muteins SEQ ID NOs: 7, 8, 9 and11 are capable of blocking the biological activity of hAng-2 in acell-based proliferation assay. In the assay, SEQ ID NOs: 7, 8, 9, 11and 16, an IgG isotype negative control and two benchmark antibodieswere added to starved HLEC. The experiment shows that LEC proliferationis blocked by SEQ ID NOs: 7, 8, 9, 11 and the benchmark antibodies 1 and2 (benchmark antibody 1: SEQ ID NOs: 17/18; benchmark antibody 2: SEQ IDNOs: 19/20 with similar range IC50 values. The IgG isotype and SEQ IDNO: 16 negative controls had no effect on cell proliferation. Data werefitted with a sigmoidal dose-response model.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

The current disclosure provides a polypeptide having binding specificityfor Ang-2, wherein the polypeptide comprises an hNGAL mutein that bindsAng-2 with detectable affinity.

In some embodiments, an hNGAL mutein binding Ang-2 with detectableaffinity may include at least one amino acid substitution of a nativecysteine residue by another amino acid, for example, a serine residue.In some other embodiments, a mutein binding Ang-2 with detectableaffinity may include one or more non-native cysteine residuessubstituting one or more amino acids of wild-type hNGAL. In a furtherparticular embodiment, an hNGAL mutein according to the disclosureincludes at least two amino acid substitutions of a native amino acid bya cysteine residue, hereby to form one or more cysteine bridges. In someembodiments, said cysteine bridge may connect at least two loop regions.The definition of these regions is used herein in accordance with Flower(Flower, 1996, supra, Flower, et al., 2000, supra) and Breustedt et al.(2005, supra).

A mutein or a composition thereof has specificity for Ang-2 as disclosedherein may have antagonist, or neutralizing or blocking activity withrespect to at least one biological activity of Ang-2.

In one aspect, the present disclosure includes various hNGAL muteinsthat bind Ang-2 with at least detectable affinity. In this sense, Ang-2is regarded as a non-natural ligand of the reference wild-type hNGAL,where “non-natural ligand” refers to a compound that does not bind towild-type human lipocalin 2 under physiological conditions. Byengineering wild-type hNGAL with one or more mutations at certainsequence positions, the present inventors have demonstrated that highaffinity and high specificity for the non-natural ligand, Ang-2, Ispossible. In some embodiments, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or even more nucleotide triplet(s) encoding certain sequence positionson wild-type I human lipocalin 2, a random mutagenesis may be carriedout through substitution at these positions by a subset of nucleotidetriplets.

Further, the muteins of the disclosure may have a mutated amino acidresidue at any one or more, including at least at any one, two, three,four, five, six, seven, eight, nine, ten, eleven or twelve, of thesequence positions corresponding to certain sequence positions of thelinear polypeptide sequence of hNGAL, such as sequence positions 28, 36,40, 41, 49, 52, 65, 68, 70, 72-74, 77, 79, 81, 87, 96, 100, 103, 106,116, 125, 126, 127, 129, 132 and 134 of the linear polypeptide sequenceof human NGAL (SEQ ID NO: 16).

A mutein of the disclosure may include the wild type (natural) aminoacid sequence of the “parental” protein scaffold (such as hNGAL) outsidethe mutated amino acid sequence positions. In some embodiments, an hNGALmutein according to the disclosure may also carry one or more amino acidmutations at a sequence position/positions as long as such a mutationdoes, at least essentially not hamper or not interfere with the bindingactivity and the folding of the mutein. Such mutations can beaccomplished very easily on DNA level using established standard methods(Sambrook, J. et al. (2001) Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Illustrative examples of alterations of the amino acid sequence areinsertions or deletions as well as amino acid substitutions. Suchsubstitutions may be conservative, i.e. an amino acid residue isreplaced with an amino acid residue of chemically similar properties, inparticular with regard to polarity as well as size. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan. On the other hand, it is also possible tointroduce non-conservative alterations in the amino acid sequence. Inaddition, instead of replacing single amino acid residues, it is alsopossible to either insert or delete one or more continuous amino acidsof the primary structure of the human lipocalin 2 as long as thesedeletions or insertion result in a stable folded/functional mutein (forexample, hNGAL muteins with truncated N- and C-terminus). In suchmutein, for instance, one or more amino acid residues are added ordeleted at the N- or C-terminus of the polypeptide. Generally such amutein may have about at least 70%, including at least about 80%, suchas at least about 85% amino acid sequence identity, with the amino acidsequence of the mature hNGAL.

The amino acid sequence of an hNGAL mutein disclosed herein has a highsequence identity to the mature hNGAL (SEQ ID NO: 16) when compared tosequence identities with other lipocalins. In this general context, theamino acid sequence of a mutein of the disclosure is at leastsubstantially similar to the amino acid sequence of the naturalwild-type hNGAL, with the proviso that possibly there are gaps (asdefined below) in an alignment that are the result of additions ordeletions of amino acids. A respective sequence of a mutein of thedisclosure, being substantially similar to the sequences of the maturehNGAL, has, in some embodiments, at least 70% identity or sequencehomology, at least 75% identity or sequence homology, at least 80%identity or sequence homology, at least 82% identity or sequencehomology, at least 85% identity or sequence homology, at least 87%identity or sequence homology, or at least 90% identity or sequencehomology including at least 95% identity or sequence homology, to thesequence of the mature hNGAL, with the proviso that the altered positionor sequence is retained and that one or more gaps are possible.

As used herein, a mutein of the disclosure “specifically binds” a target(for example, Ang-2) if it is able to discriminate between that targetand one or more reference targets, since binding specificity is not anabsolute, but a relative property. “Specific binding” can be determined,for example, in accordance with Western blots, ELISA-, RIA-, ECL-,IRMA-tests, FACS, IHC and peptide scans.

In one embodiment, the muteins of the disclosure are fused at itsN-terminus and/or its C-terminus to a fusion partner, which, in someparticular embodiments, is a protein, or a protein domain or a peptide.In some embodiments, the protein domain may extend the serum half-lifeof the mutein. In further particular embodiments, the protein domain isan Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, aCH4 domain of an immunoglobulin, an albumin binding peptide, or analbumin binding protein.

In another embodiment, the muteins of the disclosure are conjugated to acompound that extends the serum half-life of the mutein. Morepreferably, the mutein is conjugated to a compound selected from thegroup consisting of a polyalkylene glycol molecule, a hydroethylstarch,an Fc part of an immunoglobulin, a CH3 domain of an immoglobulin, a CH4domain of an immunoglobulin, an albumin binding peptide, and an albuminbinding protein.

In yet another embodiment, the current disclosure relates to a nucleicacid molecule comprising a nucleotide sequence encoding a muteindisclosed herein. The disclosure encompasses a host cell containing saidnucleic acid molecule.

A. Exemplary Muteins Specific for Ang-2

In one aspect, the present disclosure relates to novel, specific-bindinghuman lipocalin 2 (human Lcn2 or hNGAL) muteins specific for Ang-2.

One embodiment of the current disclosure relates to a mutein that iscapable of binding Ang-2 with detectable affinity, such as an affinitymeasured by a KD of about 200 nM or lower, such as about 150 nM orlower.

In one aspect, the current disclosure provides an hNGAL mutein that iscapable of binding Ang-2 with a K_(D) of about 5 nM or lower whenmeasured by Biacore T200 instrument in a Surface Plasmon Resonance (SPR)based assay essentially described in Example 6.

In some further embodiments, one or more hNGAL muteins of thisdisclosure are capable of binding Ang-2 with an affinity measured by anEC50 value of about 5 nM or lower, when measured in an ELISA assayessentially described in Example 4.

In some other embodiments, one or more hNGAL muteins of this disclosureare capable of binding Ang-2 with an affinity measured by an IC50 valueof about 5 nM or lower, when measured in a competition ELISA formatassay essentially described in Example 5.

In some other embodiments, one or more hNGAL muteins of this disclosureare capable of inhibiting or reducing lymphatic microvascularendothelial cells proliferation mediated by Ang-2 with an IC50 value ofabout 5 nM or lower in a cell-based proliferation assay essentiallydescribed in Example 9.

In some particular embodiments, an Ang-2-binding hNGAL mutein of thedisclosure is capable binding both Ang-2 and Ang-1 with detectableaffinity, such as an affinity measured by a KD of about 200 nM or lower,such as about 150 nM or lower. In some embodiments, one or more hNGALmuteins of this disclosure are crossreactive with both human Ang-1 andhuman Ang-2.

In some still further embodiments, the mutein is capable of bindingAng-1 with an affinity measured by an IC50 value of about 150 nM orlower, when measured in an ELISA assay essentially described in Example7.

In some other embodiments, one or more hNGAL muteins of this disclosureare crossreactive with both human Ang-2 and mouse Ang-2. In someembodiments, one or more such muteins are capable binding both humanAng-2 and mouse Ang-2 with detectable affinity, such as an affinitymeasured by a K_(D) of about 200 nM or lower, such as about 150 nM orlower.

In some still further embodiments, one or more such muteins are capableof binding mouse Ang-2 with an affinity measured by an IC50 value ofabout 5 nM or lower, when measured in an ELISA assay essentiallydescribed in Example 7.

In some still further embodiments, one or more such muteins are capableof blocking binding of human Ang-2 to hTie-2 and mouse Ang-2 hTie-2 withan IC50 value of about 25 nM or lower, respectively, in a competitioncell ECL format essentially described in Example 8.

In some embodiments, one or more hNGAL muteins of this disclosure arenot crossreactive with human Ang-4. In some embodiments, one or morehNGAL muteins of this disclosure are not crossreactive with mouse Ang-3.In some embodiments, one or more hNGAL muteins of this disclosure arenot crossreactive with human VEGF-A.

In this regard, the disclosure relates to a polypeptide, wherein saidpolypeptide includes an hNGAL mutein, and said hNGAL in comparison withthe linear polypeptide sequence of the mature hNGAL, comprises at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or even more, mutated amino acid residues at the sequence positions28, 36, 40, 41, 49, 52, 65, 68, 70, 72-74, 77, 79, 81, 87, 96, 100, 103,106, 116, 125, 126, 127, 129, 132 and 134, and wherein said polypeptidebinds Ang-2 with detectable affinity.

In some embodiments, an Ang-2-binding hNGAL mutein of the disclosureincludes, at any one or more of the sequence positions 36, 40, 41, 49,52, 68, 70, 72-73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132 and 134of the linear polypeptide sequence of the mature hNGAL (SEQ ID NO: 16),one or more of the following mutated amino acid residues: Leu 36→Gln,Glu, His, Val, Met or Phe; Ala 40→Val, Tyr, His or Trp; Ile 41→His, Tyr,Trp or Val; Gln 49→Gly, Ile, Val, Glu or Val; Tyr 52→Trp, His, Thr orSer; Ser 68→Gly, Asp, Gln, Glu or Ile; Leu 70→Ser, Thr, Gly, Arg, Tyr orAla; Arg 72→Gly, Ala, Trp, Thr or Glu; Lys 73→Pro, Phe, Leu. Arg, Ala orGln; Asp 77→Asn, Lys, Ser or Val; Trp 79→Thr, Arg, Ser or Asn; Arg81→Trp, His or Tyr; Asn 96→Gly, Ala, Pro, Gln or Asp; Tyr 100→Pro, Trp,Gly, Ser, Leu or Asp; Leu 103→Gly, Glu, Aso, Met or Gln; Tyr 106→Thr,Leu or Phe; Lys 125→His, Thr or Gly; Ser 127→Leu or Met; Tyr 132→Phe,Trp or Val; and Lys 134→Ala, Glu or Trp. In some embodiments, an hNGALmutein of the disclosure includes two or more, such as 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, or even more or all mutated amino acidresidues at these sequence positions of the mature hNGAL.

Additionally, an Ang-2-binding hNGAL mutein according to the disclosuremay also comprise the following substitution in comparison with thelinear polypeptide sequence of the mature hNGAL: Gln 28→His; Asn 65→Asp;Lys 74→Glu; Cys 87→Ser; Asn 116→Asp; Val 126→Met and Asn 129→Asp.

In some additional embodiments, an hNGAL mutein of the disclosure, whichbinds to Ang-2, includes the following amino acid replacements incomparison with the linear polypeptide sequence of the mature hNGAL:

-   -   (a) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Gln 49→Gly; Tyr 52→Trp;        Ser 68→Gly; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp 77→Asn; Trp        79→Thr; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr 100→Pro; Leu        103→Gly; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr 132→Phe; Lys        134→Glu;    -   (b) Gln 28→His; Leu 36→Phe; Ala 40→His; Ile 41→Arg; Gln 49→Gly;        Tyr 52→His; Ser 68→Asp; Leu 70→Thr; Arg 72→Ala; Lys 73→Phe; Asp        77→Asn; Trp 79→Arg; Arg 81→His; Cys 87→Ser; Tyr 100→Trp; Leu        103→Glu; Tyr 106→Thr; Lys 125→Thr; Ser 127→Met; Tyr 132→Trp; Lys        134→Trp;    -   (c) Gln 28→His; Leu 36→Val; Ala 40→Trp; Ile 41→Tyr; Gln 49→Ile;        Tyr 52→Thr; Ser 68→Gln; Leu 70→Gly; Arg 72→Glu; Lys 73→Gln; Asp        77→Lys; Trp 79→Ser; Arg 81→His; Cys 87→Ser; Tyr 100→Trp; Leu        103→Asp; Tyr 106→Leu; Lys 125→Gly; Ser 127→Met; Tyr 132→Val; Lys        134→Ala;    -   (d) Gln 28→His; Leu 36→Glu, Ala 40→Val; Ile 41→Glu; Gln 49→Val;        Tyr 52→Thr Ser 68→Glu; Leu 70→Arg; Arg 72→Trp; Lys 73→Leu; Asp        77→Lys; Trp 79→Asn; Arg 81→His; Cys 87→Ser; Asn 96→Ala; Tyr        100→Gly; Leu 103→Met; Tyr 106→Thr; Lys 125→Thr; Ser 127→Met; Tyr        132→Trp; Lys 134→Trp;    -   (e) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Ile 41→Trp; Gln 49→Ile;        Tyr 52→Ser; Ser 68→Ile; Leu 70→Tyr; Arg 72→Thr; Lys 73→Arg; Asp        77→Ser; Trp 79→Arg; Arg 81→Tyr; Cys 87→Ser; Asn 96→Pro; Leu        103→Asp; Tyr 106→Thr; Lys 125→His; Ser 127→Tyr; Tyr 132→Trp; Lys        134→Glu;    -   (f) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Gln 49→Glu; Tyr 52→Trp;        Asn 65→Asp; Ser 68→Gly; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp        77→Asn; Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr        100→Ser, Leu 103→Gln; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr        132→Phe; Lys 134→Glu;    -   (g) Gln 28→His; Leu 36→His; Ala 40→Tyr; Gln 49→Glu; Tyr 52→Trp;        Asn 65→Asp: Ser 68→Glu; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp        77→Asn; Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr        100→Pro; Leu 103→Asp; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr        132→Phe; Lys 134→Glu;    -   (h) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Gln 49→Gly; Tyr 52→Trp;        Asn 65→Asp; Ser 68→Glu; Leu 70→Ser; Arg 72→Gly; Lys 73→Ala; Asp        77→Asn; Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr        100→Asp; Leu 103→Gly; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr        132→Phe; Lys 134→Glu;    -   (i) Gln 28→His; Leu 36→His; Ala 40→Tyr; Gln 49→Gly; Tyr 52→Trp;        Asn 65→Asp; Ser 68→Glu; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp        77→Asn; Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr        100→Pro; Leu 103→Gly; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr        132→Phe; Lys 134→Glu;    -   (j) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Gln 49→Gly; Tyr 52→Trp;        Asn 65→Asp; Ser 68→Gly; Leu 70→Ser; Arg 72→Gly; Lys 73→Ala; Asp        77→Val; Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr        100→Pro; Leu 103→Gly; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr        132→Phe; Lys 134→Glu;    -   (k) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Gln 49→Val; Tyr 52→Trp;        Asn 65→Asp; Ser 68→Glu; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp        77→Asn; Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr        100→Leu; Leu 103→Gly; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr        132→Phe; Lys 134→Glu;    -   (l) Gln 28→His; Leu 36→Val; Ala 40→Tyr Ile 41→Tyr; Gln 49→Ile;        Tyr 52→Thr; Asn 65→Asp; Ser 68→Gln; Leu 70→Gly; Arg 72→Glu; Lys        73→Gln; Lys 74→Glu; Asp 77→Lys; Trp 79→Ser; Arg 81→His; Cys        87→Ser; Tyr 100→Trp; Leu 103→Asp; Tyr 106→Pro; Asn 116→Asp; Lys        125→Gly; Ser 127→Met; Asn 129→Asp; Tyr 132→Val; Lys 134→Ala;    -   (m) Gln 28→His; Leu 36→Val; Ala 40→Tyr; Ile 41→Tyr; Gln 49→Ile;        Tyr 52→Thr; Asn 65→Asp; Ser 68→Gln; Leu 70→Gly; Arg 72→Glu; Lys        73→Gln; Lys 74→Glu; Asp 77→Lys; Trp 79→Ser; Arg 81→His; Cys        87→Ser; Asn 96→Asp; Tyr 100→Trp; Leu 103→Asp; Tyr 106→Pro; Lys        125→Gly; Val 126→Met; Ser 127→Met; Asn 129→Asp; Tyr 132→Val; Lys        134→Ala; or    -   (n) Gln 28→His; Leu 36→Met; Ala 40→Tyr Ile 41→Asp; Gln 49→Ile;        Tyr 52→Thr; Asn 65→Asp; Ser 68→Gln; Leu 70→Gly; Arg 72→Glu; Lys        73→Gln; Asp 77→Lys; Trp 79→Ser; Arg 81→His; Cys 87→Ser; Asn        96→Gln; Tyr 100→Trp; Leu 103→Asp; Tyr 106→Pro; Lys 125→Gly; Ser        127→Met; Tyr 132→Val; Lys 134→Ala.

In the residual region, i.e. the region differing from sequencepositions 28, 36, 40, 41, 49, 52, 65, 68, 70, 72-74, 77, 79, 81, 87, 96,100, 103, 106, 116, 125, 126, 127, 129, 132 and 134, an hNGAL mutein ofthe disclosure may include the wild type (natural) amino acid sequenceoutside the mutated amino acid sequence positions.

In further particular embodiments, a mutein according to the currentdisclosure comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-14 or a functional fragment or variantthereof. In some embodiments, such fragment or variant is a structuralhomologue of a mutein defined in any one of SEQ ID NOs: 1-14.

The amino acid sequence of an Ang-2-binding hNGAL mutein of thedisclosure may have a high sequence identity, such as at least 70%, atleast 75%, at least 80%, at least 82%, at least 85%, at least 87%, atleast 90% identity, including at least 95% identity, to a sequenceselected from the group consisting of SEQ ID NOs: 1-14.

In some still embodiments, an hNGAL mutein crossreactive with or bindingto human Ang-1, human Ang-2 and/or mouse Ang-2 according to thedisclosure comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-14 and functional fragments or variantsthereof.

The disclosure also includes structural homologues of an hNGAL muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 1-14, which structural homologues have an amino acid sequencehomology or sequence identity of more than about 60%, preferably morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 92% and most preferably more than 95% inrelation to said hNGAL mutein.

An Ang-2-binding hNGAL mutein according to the present disclosure can beobtained by means of mutagenesis of a naturally occurring form of humanlipocalin 2. In some embodiments of the mutagenesis, a substitution (orreplacement) is a conservative substitution. Nevertheless, anysubstitution—including non-conservative substitution or one or more fromthe exemplary substitutions below—is envisaged as long as the muteinretains its capability to bind to Ang-2, and/or it has an identity tothe then substituted sequence in that it is at least 60%, such as atleast 65%, at least 70%, at least 75%, at least 80%, at least 85% orhigher identity to the amino acid sequence of the mature human lipocalin2 (SWISS-PROT Data Bank Accession Number P80188).

The present disclosure also relates to a pharmaceutical composition thatincludes at least one Ang-2-binding hNGAL mutein disclosed herein, orconjugate or fusion protein thereof as described herein, and optionally,a pharmaceutically acceptable excipient.

Accordingly, the Ang-2-binding hNGAL muteins of the disclosure can beformulated into compositions using pharmaceutically acceptableingredients as well as established methods of preparation (Gennaro andGennaro (2000) Remington: The Science and Practice of Pharmacy, 20thEd., Lippincott Williams & Wilkins, Philadelphia, Pa.). To prepare thepharmaceutical compositions, pharmaceutically inert inorganic or organicexcipients can be used.

B. Applications of Muteins Specific for Ang-2

Recently, using an ANG-2 knockout mouse model, Yancopoulos' groupreported that ANG-2 is required for postnatal angiogenesis (Gale, N. W.,et al., Dev. Cell 3 (2002) 411-23). They showed that the developmentallyprogrammed regression of the hyaloid vasculature in the eye does notoccur in the ANG-2 knockout mice and their retinal blood vessels fail tosprout out from the central retinal artery (Gale, N. W., et al., Dev.Cell 3 (2002) 411-23). They also found that deletion of ANG-2 results inprofound defects in the patterning and function of the lymphaticvasculature (Gale, N. W., et al., Dev. Cell 3 (2002) 411-23). Inaddition, effective anti-Ang-2 therapy is thought to be of benefit intreating diseases such as cancer, in which progression is dependent onaberrant angiogenesis where blocking the process can lead to preventionof disease advancement (Folkman, J., Nature Medicine. 1, (1995) 27-31).Moreover, Ang-2 expression has been shown to correlate with the severityof various inflammatory and/or infectious diseases (see, e.g., Siner etal., 2009, Shock 31:348-353; Yeo et al., 2008, Proc. Natl. Acad. Sci.(USA): 105: 17097-17102).

Numerous possible applications for the muteins with binding-affinity forAng-2 of the disclosure, therefore, exist in medicine, for example, inophthalmology and oncology. In one further aspect, the disclosurerelates to the use of such a mutein disclosed herein for detecting Ang-2in a sample as well as a respective method of diagnosis.

The present disclosure also involves the use of one or more muteins withbinding-affinity for Ang-2 as described for complex formation withAng-2.

Therefore, in another aspect of the disclosure, the disclosed muteinsare used for the detection of Ang-2. Such use may include the steps ofcontacting one or more said muteins, under suitable conditions, with asample suspected of containing Ang-2, thereby allowing formation of acomplex between the muteins and Ang-2, and detecting the complex by asuitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is surface plasmon resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins disclosed herein may also be used for the separation ofAng-2. Such use may include the steps of contacting one or more saidmuteins, under suitable conditions, with a sample supposed to containAng-2, thereby allowing formation of a complex between the muteins andAng-2, and separating the complex from the sample.

In the use of the disclosed muteins for the detection of Ang-2 as wellas the separation of Ang-2, the muteins and/or Ang-2 or a domain orfragment thereof may be immobilized on a suitable solid phase.

Accordingly, the presence or absence of a molecule such as Ang-2, e.g.,in a sample, as well as its concentration or level may be determined.

The muteins of the present disclosure, therefore, may be used to detectand/or measure Ang-2 in a sample, e.g., for diagnostic purposes. Forexample, the muteins may be used to diagnose a condition or diseasecharacterized by aberrant expression (e.g., over-expression,under-expression, lack of expression, etc.) of Ang-2. Exemplarydiagnostic assays for Ang-2 may comprise, e.g., contacting a sample,obtained from a patient, with the muteins, wherein the muteins arelabeled with a detectable label or reporter molecule. Alternatively,unlabeled the muteins can be used in diagnostic applications incombination with a secondary molecule which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemlluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase, 3-galactosidase,horseradish peroxidase, or luciferase. Specific exemplary assays thatcan be used to detect or measure Ang-2 in a sample include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence-activated cell sorting (FACS).

In still another aspect, the present disclosure features a diagnostic oranalytical kit comprising a mutein with binding-affinity for Ang-2according to the disclosure.

In a further aspect, the disclosure also encompasses the use ofdisclosed Ang-2-binding hNGAL muteins or combinations comprising suchmuteins described herein for the manufacture of a pharmaceuticalcomposition. The pharmaceutical composition thus obtained may be suitedfor the treatment, prevention and/or amelioration of a disease ordisorder associated with deregulated angiogenesis, such as cancer,ocular neovascular diseases (such as retinopathies), arthritis, andpsoriasis. The pharmaceutical composition may be used as monotherapy oras combination therapy. Accordingly, the disclosure also providesAng-2-binding hNGAL muteins for the treatment of a disease or disorderassociated with deregulated angiogenesis such as cancer, ocularneovascular diseases (such as retinopathies), arthritis, and psoriasis.

In addition to their use in diagnostics, in yet another aspect, thedisclosure encompasses the use of such a mutein of the disclosure or acomposition or a combination comprising such mutein for the binding ofAng-2 in a subject and/or inhibiting or reducing angiogenesis in asubject. In some embodiments, such subject may suffer from diseases ordisorders associated with deregulated angiogenesis such as cancer,ocular neovascular diseases (such as retinopathies), arthritis, andpsoriasis.

In still another aspect, the present disclosure features a method ofbinding Ang-2 in a subject, comprising administering to said subject aneffective amount of one or more muteins with binding-affinity for Ang-2of the disclosure or of one or more compositions or combinationscomprising such a mutein.

In still another aspect, the present disclosure involves a method forinhibiting or reducing angiogenesis in a subject, comprisingadministering to said subject an effective amount of one or more muteinswith binding-affinity for Ang-2 of the disclosure or of one or morecompositions or combinations comprising such a mutein. In someembodiments, such subject may suffer from diseases or disordersassociated with deregulated angiogenesis such as cancer, ocularneovascular diseases (such as retinopathies), arthritis, and psoriasis.

The muteins of the disclosure or compositions or combinations comprisingsuch muteins are useful, inter alia, for the treatment, preventionand/or amelioration of any disease or disorder associated with Ang-2activity, including diseases or disorders associated with deregulatedangiogenesis.

For example, the muteins or compositions or combinations comprising suchmuteins of the disclosure may be used to inhibit or reduce tumor growthcharacterized by undesired angiogenesis in a subject, such as withrespect to primary and/or metastatic tumors arising in the brain andmeninges, oropharynx, lung and bronchial tree, gastrointestinal tract,male and female reproductive tract, muscle, bone, skin and appendages,connective tissue, spleen, immune system, blood forming cells and bonemarrow, liver and urinary tract, and special sensory organs such as theeye. In certain embodiments, the antibodies and antigen-bindingfragments of the disclosure are used to treat one or more of thefollowing cancers: renal cell carcinoma, pancreatic carcinoma, breastcancer, prostate cancer, malignant gliomas, osteosarcoma, colorectalcancer, malignant mesothelioma, multiple myeloma, ovarian cancer, smallcell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroidcancer, or melanoma.

In some other embodiments, the muteins or compositions or combinationscomprising such muteins of the present disclosure may also be useful forthe treatment, prevention and/or amelioration of one or more eyedisorders such as retinopathy. Exemplary eye disorders that can betreated, prevented and/or ameliorated with or by the muteins orcompositions comprising such muteins include, e.g., age-related maculardegeneration, diabetic retinopathy, and other eye disorders associatedwith choroidal neovascularization, vascular leak, retinal edema andinflammation. Additionally, the muteins may be administered as anadjuvant to glaucoma surgery to prevent early hem- and lymphangiogenesisand macrophage recruitment to the filtering bleb after glaucoma surgery,and improve clinical outcome.

In some additional embodiments, the muteins or compositions orcombinations comprising such muteins are used to treat, prevent orameliorate vascular diseases such as hypertension, diabetes (includingnon-insulin dependent diabetes mellitus), psoriasis, arthritis(including rheumatoid arthritis), asthma, sepsis, kidney disease andedema. In some further embodiments, these diseases or disorders areassociated with injury, stroke or tumor.

Furthermore, the muteins or compositions or combinations comprising suchmuteins disclosed herein can be used to treat, prevent or ameliorate oneor more inflammatory or infectious diseases. Exemplary infectiousdiseases that can be treated, prevented or ameliorated by administrationof the anti-Ang-2 muteins of the disclosure or compositions comprisingsuch muteins include, but are not limited to, malaria (Plasmodiumfalciparum infection), viral hemorrhagic fevers (e.g., dengue fever),rickettsial infection, toxic shock syndrome, sepsis, hepatitis C,Bartonella bacilliformis infection, leishmaniasis, mycobacterialinfection, and Epstein-Barr virus infection.

The current disclosure also contemplates the use of the muteinsdisclosed herein or compositions or combinations comprising such muteinsfor the preparation of a medicament for the prevention of metastasis orthe treatment of cancer or vascular diseases.

The Ang-2-binding hNGAL muteins according to the disclosure orcompositions or combinations comprising such muteins can be administeredvia any parenteral or non-parenteral (e.g. enteral) route that istherapeutically effective. A therapeutically effective route providesfor delivery of an agent to a desired compartment, system, or location.For example, a therapeutically effective route is one through which anagent can be administered to provide at the desired site of action anamount of the agent sufficient to effectuate a beneficial or desiredclinical result.

C. Combination of Ang-2-Binding Mutein with One or More Anti-AngiogenicAgents

Angiogenesis requires the binding of signaling molecules, such asvascular endothelial growth factor (VEGF), to receptors on the surfaceof normal endothelial cells. When VEGF and other endothelial growthfactors bind to their receptors on endothelial cells, signals withinthese cells are initiated that promote the growth and survival of newblood vessels. One approach to developing an effective anti-angiogenictreatment modality has been to combine agents that act on differenttargets involved in angiogenesis, preferably targets that act on wellisolated signaling pathways.

The present disclosure, therefore, encompasses the use of an hNGALmutein of the disclosure specific for Ang-2 in combination with one ormore anti-angiogenic agents. As used here, an “anti-angiogenic agent”means any substance is capable of inhibiting or interfering with thebinding of one of such signaling molecules to its receptor. In someembodiments, the anti-angiogenic agent is capable of blocking orcontributes to block the one of signals that promotes the growth andsurvival of new blood vessels.

In some particular embodiments, the anti-angiogenic agents comprises (i)antagonists of Ang-1, Ang-2, Ang-3, Ang-4 and/or Tie-2; (ii) antagonistsof Flt1, KDR, Flt4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PIGF and/orEG-VEGF; (iii) delta like ligand 4 (DLL4, a vascular-specific Notchligand) antagonists, (iv) epidermal growth factor receptor (EGFR)antagonists and (v) cytokine inhibitors.

In some still further embodiments, the Ang-2 antagonist may be an hNGALmutein of the disclosure, an anti-Ang-2 antibody (see U.S. Pat. No.8,133,979; incorporated herein by reference in its entirety), peptibody(see U.S. Pat. No. 8,129,331; incorporated herein by reference in itsentirety), or CovX-body (such as CVX-060, see U.S. Pat. No. 7,521,425;incorporated herein by reference in its entirety). In some furtherembodiments, the DLL4 antagonist may be an anti-DLL4 antibody (e.g., ananti-DLL4 antibody disclosed in U.S. Patent Application No. 2009/0142354such as REGN421 and etc.). In some further embodiments, the EGFRantagonist may be an anti-EGFR antibody or small molecule inhibitor ofEGFR activity. Other anti-angiogenic agents that may be beneficiallyadministered in combination with the anti-Ang-2 hNGAL muteins of thedisclosure include cytokine inhibitors, including small-moleculecytokine inhibitors and antibodies that bind to cytokines such as IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17,IL-18, and/or to their respective receptors.

In this regard, the present disclosure also includes therapeuticcombinations comprising any of the anti-Ang-2 hNGAL muteins mentionedherein and an anti-angiogenic agent such as an antagonist of one or moreof VEGF, DLL4, EGFR, or any of the aforementioned cytokines, wherein theantagonist may be an aptamer, an antisense molecule, a ribozyme, ansiRNA, a peptibody, a nanobody, an antibody, an antibody fragment (e.g.,Fab fragment; F(ab′)₂ fragment; Fd fragment; Fv fragment; scFv; dAbfragment; an engineered molecule (such as diabody, triabody, tetrabody,minibody and minimal recognition unit); an antiviral, an antibiotic, ananalgesic, a corticosteroids and/or an nonsteroidal anti-inflammatorydrug (NSAID).

In some embodiments, said engineered molecule may be an EGF-like domain,a Kringle-domain, a fibronectin type I domain, a fibronectin type IIdomain, a fibronectin type III domain, a PAN domain, a G1a domain, aSRCR domain, a Kunitz/Bovine pancreatic trypsin inhibitor domain,tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil(P-type) domain, a von Willebrand factor type C domain, anAnaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat,LDL-receptor class A domain, a Sushi domain, a Link domain, aThrombospondin type I domain, an immunoglobulin domain or a animmunoglobulin-like domain (for example, domain antibodies or camelheavy chain antibodies), a C-type lectin domain, a MAM domain, a vonWillebrand factor type A domain, a Somatomedin B domain, a WAP-type fourdisulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain, a“kappabody” (III. et al. “Design and construction of a hybridimmunoglobulin domain with properties of both heavy and light chainvariable regions” Protein Eng 10:949-57 (1997)), a “minibody” (Martin etal. “The affinity-selection of a minibody polypeptide inhibitor of humaninterleukin-6” EMBO J 13:5303-9 (1994)), a “diabody” (Holliger et al.“‘Diabodies’: small bivalent and bispecific antibody fragments” PNAS USA90:6444-6448 (1993)), a “janusin” (Traunecker et al. “Bispecific singlechain molecules (Janusins) target cytotoxic lymphocytes on HIV infectedcells” EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: newmolecular design for bispecific reagents” Int J Cancer Suppl 7:51-52(1992), a nanobody, an adnectin, a tetranectin, a microbody, an affilin,an affibody an ankyrin, a crystallin, a knottin, ubiquitin, azinc-finger protein, an autofluorescent protein, an ankyrin or ankyrinrepeat protein or a leucine-rich repeat protein, or an avimer(Silverman, Lu Q, Bakker A, To W, Duguay A, Alba B M, Smith R, Rivas A,Li P, Le H, Whitehorn E, Moore K W, Swimmer C, Perlroth V, Vogt M,Kolkman J, Stemmer W P 2005, Nat Biotech, December; 23(12):1556-61,E-Publication in Nat Biotech. 2005 Nov. 20 edition).

When combined with one or more additional agents of the disclosure, theanti-Ang-2 hNGAL muteins may be administered prior to, simultaneous with(e.g., in the same formulation or in separate formulations), orsubsequent to the administration of the other agent(s). The hNGAL muteinand the anti-angiogenic agent may be administered in combination,including concurrently, concomitantly or in series. In some embodiments,the combinations of the disclosure, the hNGAL muteins and theanti-angiogenic agents, may be included in a single composition that maybe administered. The composition may include an effective amount of thehNGAL mutein and the anti-angiogenic agent as active ingredients, inassociation with at least one pharmaceutically acceptable adjuvant,diluent or carrier. In this regard, the combinations of the disclosurecan be formulated into compositions using pharmaceutically acceptableingredients as well as established methods of preparation (Gennaro andGennaro (2000) Remington: The Science and Practice of Pharmacy, 20thEd., Lippincott Williams & Wilkins, Philadelphia, Pa.). To prepare thepharmaceutical compositions, pharmaceutically inert inorganic or organicexcipients can be used.

The hNGAL mutein and the anti-angiogenic agent may also be administeredindependent from each other, including at individual intervals atindependent points of time. The combinations of the hNGAL mutein and theanti-angiogenic agent may be provided in various forms and in anyorientation.

The anti-Ang-2 hNGAL muteins of the disclosure and combinations thereofmay also be administered as part of a treatment regimen that alsoincludes radiation treatment and/or conventional chemotherapy.

In some particular embodiments, the anti-angiogenic agent is anantagonist of any component of the VEGF/VEGF receptor systems andAngiopoietin/Tie-2 receptor system; that is any one of Flt1, KDR, Flt4,VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PIGF, EG-VEGF, Ang-1, Ang-2,Ang-3, Ang-4 or Tie-2. The VEGF-VEGFR pathway and the Tie-2 pathwayshould be considered as two independent mediators essential for theprocess of in vivo angiogenesis (Siemeister, G., et al., Cancer Res.59:3 (1999) 3185-91; Jendreyko, N., et al., Journal of BiologicalChemistry, 278:47812-47819 (2003); Jendreyko, N., et al., PNAS,102:8293-8298 (2005)).

In some still further embodiments, the anti-angiogenic agent is a VEGFinhibitor. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGF-E, placental growth factor (PIGF) and endocrine gland-derived VEGF(EG-VEGF). Members of the VEGF family are known to bind with differentaffinities to three related receptor tyrosine kinases: VEGFR1 (thefms-like tyrosine kinase receptor, Flt1), VEGFR2 (the kinase insertdomain-containing receptor, KDR), and VEGFR3 (another fms-like tyrosinekinase receptor, Flt4).

A “VEGF inhibitor” as used herein means any substance that decreasessignaling by the VEGF-VEGFR pathway. Therefore, VEGF inhibitor mayinhibit or interfere with any member of the VEGF family and/or any oneof the receptor tyrosine kinases. VEGF inhibitors can be, to name just afew examples, small molecules, peptides, polypeptides, proteins,including more specifically antibodies, including anti-VEGF antibodies,anti-VEGFR antibodies, intrabodies, maxibodies, minibodies, diabodies,Fc fusion proteins such as peptibodies, receptibodies, soluble VEGFreceptor proteins and fragments, and a variety of others. Many VEGFinhibitors work by binding to VEGF or to a VEGF receptor. Others workmore indirectly by binding to factors that bind to VEGF or to a VEGFreceptor or to other components of the VEGF signaling pathway. Stillother VEGF inhibitors act by altering regulatory posttranslationalmodifications that modulate VEGF-VEGFR pathway signaling. VEGFinhibitors in accordance with the disclosure also may act through moreindirect mechanisms. Whatever the mechanism involved, as used herein, aVEGF inhibitor decreases the effective activity of the VEGF-VEGFRsignaling pathway in a given circumstance over what it would be in thesame circumstance in the absence of the inhibitor.

In some particular embodiments, the VEGF inhibitor may be a VEGF-Trap(see, e.g., U.S. Pat. No. 7,087,411; incorporated herein by reference inits entirety), an anti-VEGF antibody (e.g., bevacizumab), a smallmolecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenibor pazopanib), an anti-VEGF tear lipocalin mutein (see, e.g. PCTapplication PCT/EP2007/057971, incorporated herein by reference in itsentirety) or anti-VEGFR tear lipocalin mutein (see, e.g. PCT applicationPCT/EP2007/057971, Incorporated herein by reference in its entirety).

In some embodiments, the VEGF inhibitor, as the anti-angiogenic agent,is selected from the group consisting of VEGF-Trap (see, e.g., U.S. Pat.No. 7,087,411; incorporated herein by reference in its entirety),bevacizumab (Avastin®), sorafenib (Nexavar®), sunitinib (Sutent®) andpazopanib (Votrient®).

In some additional embodiments, the VEGF inhibitor may be an antagonistof VEGF-A, such as an anti-VEGF-A antibody (e.g., bevacizumab). In someother embodiments, the VEGF-A antagonist may be a lipocalin mutein thathas binding specificity for VEGF-A (see, e.g. PCT applicationPCT/EP2007/057971; incorporated herein by reference in its entirety).

In some additional embodiments, the VEGF inhibitor may be an antagonistof VEGF-C, such as an anti-VEGF-C antibody (see, e.g., U.S. Pat. Nos.6,403,088 and 8,486,397; incorporated herein by reference in theirentirety). In some other embodiments, the VEGF-C antagonist may be alipocalin mutein that has binding specificity for VEGF-C.

In some embodiments, an hNGAL mutein of the disclosure specific forAng-2 in combination with more than one anti-angiogenic agents, such astwo, wherein one agent is a VEGF-A antagonist mentioned herein and thesecond agent is a VEGF-C antagonist mentioned herein.

In some embodiments, the present disclosure encompasses the use of (i)an hNGAL mutein of the disclosure specific for Ang-2 and (ii) one ormore anti-angiogenic agents, for inhibiting deregulated angiogenesis ina subject. Such use includes a step of administering to a subject aneffective amount of (i) an hNGAL mutein of the disclosure specific forAng-2 and (ii) one or more anti-angiogenic agents.

Similarly, the present disclosure discloses the use of (i) an hNGALmutein of the disclosure specific for Ang-2 and (ii) one or moreanti-angiogenic agents for the treatment, prevention or alleviation ofdiseases or disorders associated with deregulated angiogenesis in asubject in some further embodiments, the diseases or disordersassociated with deregulated angiogenesis include cancer, ocularneovascular diseases (such as retinopathies), arthritis, and psoriasis.In some embodiments, one anti-anglogenic agent is a VEGF-A antagonistmentioned herein and the second anti-angiogenic agent is a VEGF-Cantagonist mentioned herein.

Further details on hNGAL muteins with a detectable affinity for Ang-2can be found in Section A of the current disclosure.

In a particularly preferred embodiment, an hNGAL mutein that is specificfor Ang-2 is selected from the group consisting of SEQ ID NOs: 1-14 andfunctional fragments or variants thereof. In some embodiments, suchfragments or variants are structural homologues of a mutein defined inany one of SEQ ID NOs: 1-14.

The present disclosure also relates to a pharmaceutical compositioncomprising at least one of the following: (i) an hNGAL mutein of thedisclosure specific for Ang-2 and (ii) one or more anti-angiogenicagents, which composition can be used in for inhibiting deregulatedangiogenesis. In some embodiments, one anti-angiogenic agent is a VEGF-Aantagonist mentioned herein and the second anti-angiogenic agent is aVEGF-C antagonist mentioned herein.

In this regard, the combinations of the disclosure can be formulatedinto compositions using pharmaceutically acceptable ingredients as wellas established methods of preparation (Gennaro and Gennaro (2000)Remington: The Science and Practice of Pharmacy, 20th Ed., LippincottWilliams & Wilkins, Philadelphia, Pa.). To prepare the pharmaceuticalcompositions, pharmaceutically inert inorganic or organic excipients canbe used.

In still another aspect, the present disclosure features a method oftreating, preventing or ameliorating diseases or disorders associatedwith deregulated angiogenesis in a subject, comprising administering tosaid subject an effective amount of a composition that comprises atleast the following: (i) a mutein of hNGAL that has a detectableaffinity to Ang-2 and (ii) one or more anti-angiogenic agents. In somefurther embodiments, the diseases or disorders associated withderegulated angiogenesis include cancer, ocular neovascular diseases(such as retinopathies), arthritis, and psoriasis. In some embodiments,one anti-angiogenic agent is a VEGF-A antagonist mentioned herein andthe second anti-angiogenic agent is a VEGF-C antagonist mentionedherein.

In still another aspect, the present disclosure involves a method ofinhibiting or reducing deregulated angiogenesis in a subject comprisingadministering to said subject an effective amount of a composition thatcomprises at least the following: (i) a mutein of hNGAL that has adetectable affinity to Ang-2 and (ii) one or more anti-angiogenicagents. In some embodiments, one anti-angiogenic agent is a VEGF-Aantagonist mentioned herein and the second anti-angiogenic agent is aVEGF-C antagonist mentioned herein.

D. Muteins of the Disclosure

When used herein in the context of the muteins of the present disclosurethat bind to Ang-2, the term “specific for” includes that the mutein isdirected against, binds to, or reacts with Ang-2. Thus, being directedto, binding to or reacting with includes that the mutein specificallybinds to Ang-2. The term “specifically” in this context means that themutein reacts with an Ang-2, as described herein, but essentially notwith another target. Whether the mutein specifically reacts as definedherein above can easily be tested, inter alia, by comparing the reactionof a hNGAL mutein of the present disclosure with Ang-2 and the reactionof said mutein with (an) other target(s). “Specific binding” can also bedetermined, for example, in accordance with Western blots, ELISA-, RIA-,ECL-, IRMA-tests, FACS, IHC and peptide scans.

The amino acid sequence of a mutein according to the disclosure has ahigh sequence identity to human lipocalin 2 when compared to sequenceidentities with another lipocalin (see also above). In this generalcontext the amino acid sequence of a mutein of the combination accordingto the disclosure is at least substantially similar to the amino acidsequence of the corresponding lipocalin (the wild-type hNGAL). Arespective sequence of a mutein of the combination according to thedisclosure, being substantially similar to the sequence of mature hNGAL,such as at at least 65%, at least 70%, at least 75%, at least 80%, atleast 82%, at least 85%, at least 87%, at least 90% identity, includingat least 95% identity to the sequence of mature hNGAL. In this regard, amutein of the disclosure of course may contain, in comparisonsubstitutions as described herein which renders the mutein capable ofbinding to Ang-2. Typically a mutein of hNGAL includes one or moremutations—relative to the native sequence of hNGAL—of amino acids in thefour loops at the open end of the ligand binding site of hNGAL. Asexplained above, these regions are essential in determining the bindingspecificity of a mutein for Ang-2. A mutein derived hNGAL or a homologuethereof, may have one, two, three, four or more mutated amino acidresidues at any sequence position in the N-terminal region and/or in thethree peptide loops BC, DE, and FG arranged at the end of the β-barrelstructure that is located opposite to the natural binding pocket.

A mutein according to the disclosure includes one or more, such as two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen oreven twenty substitutions in comparison to the corresponding nativehNGAL alin, provided that such a mutein should be capable of binding toAng-2. For example, a mutein can have a substitution at a positioncorresponding to a distinct position (i.e. at a corresponding position)of hNGAL. In some embodiments a mutein of the combination according tothe disclosure includes at least two amino acid substitutions, including2, 3, 4, 5, or even more, amino acid substitutions of a native aminoacid by an arginine residue. Accordingly, the nucleic acid of a protein‘reference’ scaffold as described herein is subject to mutagenesis withthe aim of generating a mutein which is capable of binding to Ang-2.

Also, a mutein of the present disclosure can comprise a heterologousamino acid sequence at its N- or C-Terminus, preferably C-terminus, suchas a Strep-tag, e.g., Strep II tag without affecting the biologicalactivity (binding to its target e.g. Ang-2) of the mutein.

Specifically, in order to determine whether an amino acid residue of theamino acid sequence of a mutein different from wild-type hNGALcorresponds to a certain position in the amino acid sequence ofwild-type hNGAL, a skilled artisan can use means and methods well-knownin the art, e.g., alignments, either manually or by using computerprograms such as BLAST2.0, which stands for Basic Local Alignment SearchTool or ClustalW or any other suitable program which is suitable togenerate sequence alignments. Accordingly, wild-type hNGAL can serve as“subject sequence” or “reference sequence”, while the amino acidsequence of a mutein different from the wild-type hNGAL described hereinserves as “query sequence”. The terms “reference sequence” and “wildtype sequence” are used interchangeably herein.

In some embodiments a substitution (or replacement) is a conservativesubstitution. Nevertheless, any substitution—including non-conservativesubstitution or one or more from the exemplary substitutions listedbelow—is envisaged as long as the mutein retains its capability to bindAng-2, and/or it has an identity to the then substituted sequence inthat it is at least 60%. such as at least 65%, at least 70%, at least75%, at least 80%, at least 85% or higher identical to the “original”sequence.

Conservative substitutions are generally the following substitutions,listed according to the amino acid to be mutated, each followed by oneor more replacement(s) that can be taken to be conservative: Ala→Gly,Ser, Val; Arg→Lys; Asn→Gln, His; Asp→Glu; Cys→Ser; Gln→Asn; Glu→Asp;Gly→Ala; His→Arg, Asn, Gln; Ile→Leu, Val; Leu→Ile, Val; Lys→Arg, Gln,Glu; Met→Leu, Tyr, Ile; Phe→Met, Leu, Tyr; Ser→Thr; Thr→Ser; Trp→Tyr;Tyr→Trp, Phe; Val→Ile, Leu. Other substitutions are also permissible andcan be determined empirically or in accord with other known conservativeor non-conservative substitutions. As a further orientation, thefollowing eight groups each contain amino acids that can typically betaken to define conservative substitutions for one another:

a. Alanine (Ala), Glycine (Gly);b. Aspartic acid (Asp), Glutamic acid (Glu);c. Asparagine (Asn), Glutamine (Gln);d. Arginine (Arg), Lysine (Lys);e. Isoleucine (Ile), Leucine (Leu). Methionine (Met), Valine (Val):f. Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp);g. Serine (Ser), Threonine (Thr); andh. Cysteine (Cys), Methionine (Met)

If such substitutions result in a change in biological activity, thenmore substantial changes, such as the following, or as further describedbelow in reference to amino acid classes, may be introduced and theproducts screened for a desired characteristic. Examples of such moresubstantial changes are: Ala→Leu, Ile; Arg→Gln; Asn→Asp, Lys, Arg→His:Asp→Asn; Cys→Ala; Gln→Glu; Glu→Gln; His→Lys; Ile→Met, Ala, Phe; Leu→Ala,Met, Norleucine; Lys→Asn; Met→Phe; Phe→Val, Ile, Ala; Trp→Phe; Tyr→Thr,Ser; Val→Met, Phe, Ala.

Substantial modifications in the biological properties of hNGAL areaccomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties: (1) hydrophobic: norleucine, methionine, alanine, valine,leucine, iso-leucine; (2) neutral hydrophilic: cysteine, serine,threonine; (3) acidic: asparitic acid, glutamic acid; (4) basic:asparagine, glutamine, histidine, lysine, arginine; (5) residues thatinfluence chain orientation: glycine, proline; and (6) aromatic:tryptophan, tyrosine, phenylalanine.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of hNGAL also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to improve its stability.

Any mutation, including an insertion as discussed above, can beaccomplished very easily on the nucleic acid, e.g. DNA level usingestablished standard methods. Illustrative examples of alterations ofthe amino acid sequence are insertions or deletions as well as aminoacid substitutions. Such substitutions may be conservative, i.e. anamino acid residue is replaced with an amino acid residue of chemicallysimilar properties, in particular with regard to polarity as well assize. Examples of conservative substitutions are the replacements amongthe members of the following groups: 1) alanine, serine, and threonine;2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) iso-leucine, leucine, methionine, and valine;and 6) phenylalanine, tyrosine, and tryptophan. On the other hand, it isalso possible to introduce non-conservative alterations in the aminoacid sequence. In addition, instead of replacing single amino acidresidues, it is also possible to either insert or delete one or morecontinuous amino adds of the primary structure of hNGAL as long as thesedeletions or insertion result in a stable folded/functional mutein.

Modifications of the amino acid sequence include directed mutagenesis ofsingle amino acid positions in order to simplify sub-cloning of themutated hNGAL gene or its parts by incorporating cleavage sites forcertain restriction enzymes. In addition, these mutations can also beincorporated to further improve the affinity of a mutein for a giventarget such as Ang-2. Furthermore, mutations can be introduced in orderto modulate certain characteristics of the mutein such as to improvefolding stability, serum stability, protein resistance or watersolubility or to reduce aggregation tendency, if necessary. For example,naturally occurring cysteine residues may be mutated to other aminoacids to prevent disulphide bridge formation. It is also possible todeliberately mutate other amino acid sequence position to cysteine inorder to introduce new reactive groups, for example for the conjugationto other compounds, such as polyethylene glycol (PEG), hydroxyethylstarch (HES), biotin, peptides or proteins, or for the formation ofnon-naturally occurring disulphide linkages. The generated thiol moietymay be used to PEGylate or HESylate the mutein, for example, in order toincrease the serum half-life of a respective mutein.

It is also possible to mutate other amino acid sequence positions tocysteine in order to introduce new reactive groups, for example, for theconjugation to other compounds, such as polyethylene glycol (PEG),hydroxyethyl starch (HES), biotin, peptides or proteins, or for theformation of non-naturally occurring disulphide linkages.

In some embodiments, if one of the above moieties is conjugated to amutein of the disclosure, conjugation to an amino acid side chain can beadvantageous. Suitable amino acid side chains may occur naturally in theamino acid sequence of hNGAL or may be introduced by mutagenesis. Incase a suitable binding site is introduced via mutagenesis, onepossibility is the replacement of an amino acid at the appropriateposition by a cysteine residue.

With respect to a mutein of human lipocalin 2, exemplary possibilitiesof such a mutation to introduce a cysteine residue into the amino acidsequence of a lipocalin including human lipocalin 2 mutein to includethe introduction of a cysteine (Cys) residue at at least at one of thesequence positions that correspond to sequence positions 14, 21, 60, 84,88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of humanNGAL. In some embodiments where a human lipocalin 2 mutein of thedisclosure has a sequence in which, in comparison to the sequence of theSWISS-PROT/UniProt Data Bank Accession Number P80188, a cysteine hasbeen replaced by another amino acid residue, the corresponding cysteinemay be reintroduced into the sequence. As an illustrative example, acysteine residue at amino acid position 87 may be introduced in such acase by reverting to a cysteine as originally present in the sequence ofSWISS-PROT accession No. P80188. The generated thiol moiety at the sideof any of the amino acid positions 14, 21, 60, 84, 88, 116, 141, 145,143, 146 and/or 158 may be used to PEGylate or HESylate the mutein, forexample, in order to increase the serum half-life of a respective humanlipocalin 2 mutein.

In another embodiment, in order to provide suitable amino acid sidechains for conjugating one of the above compounds to a mutein accordingto the present disclosure, artificial amino acids may be introduced bymutagenesis. Generally, such artificial amino acids are designed to bemore reactive and thus to facilitate the conjugation to the desiredcompound. One example of such an artificial amino acid that may beintroduced via an artificial tRNA is para-acetyl-phenylalanine.

In some embodiments, a mutein of the disclosure may be fused at itsN-terminus or its C-terminus to a protein, a protein domain or apeptide, for instance, a signal sequence and/or an affinity tag.

Affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T. G. M.et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag,the His₆-tag or the HA-tag or proteins such as glutathione-S-transferasealso allow easy detection and/or purification of recombinant proteinsare further examples of suitable fusion partners. Finally, proteins withchromogenic or fluorescent properties such as the green fluorescentprotein (GFP) or the yellow fluorescent protein (YFP) are suitablefusion partners for muteins of the disclosure as well.

In general, it is possible to label the muteins of the disclosure withany appropriate chemical substance or enzyme, which directly orindirectly generates a detectable compound or signal in a chemical,physical, optical, or enzymatic reaction. An example for a physicalreaction and at the same time optical reaction/marker is the emission offluorescence upon irradiation or the emission of X-rays when using aradioactive label. Alkaline phosphatase, horseradish peroxidase and3-galactosidase are examples of enzyme labels (and at the same timeoptical labels) which catalyze the formation of chromogenic reactionproducts. In general, all labels commonly used for antibodies (exceptthose exclusively used with the sugar moiety in the Fc part ofimmunoglobulins) can also be used for conjugation to the muteins of thedisclosure. The muteins of the disclosure may also be conjugated withany suitable therapeutically active agent, e.g., for the targeteddelivery of such agents to a given cell, tissue or organ or for theselective targeting of cells, e.g., of tumor cells without affecting thesurrounding normal cells. Examples of such therapeutically active agentsinclude radionuclides, toxins, small organic molecules, and therapeuticpeptides (such as peptides acting as agonists/antagonists of a cellsurface receptor or peptides competing for a protein binding site on agiven cellular target). The muteins of the disclosure may, however, alsobe conjugated with therapeutically active nucleic acids such asantisense nucleic acid molecules, small interfering RNAs, micro RNAs orribozymes. Such conjugates can be produced by methods well known in theart.

As Indicated above, a mutein of the disclosure may in some embodimentsbe conjugated to a moiety that extends the serum half-life of the mutein(in this regard see also PCT publication WO 2006/56464 where suchconjugation strategies are described with references to muteins of humanneutrophile gelatinase-associated lipocalin with binding affinity forCTLA-4). The moiety that extends the serum half-life may be apolyalkylene glycol molecule, hydroxyethyl starch, fatty acid molecules,such as palmitic acid (Vajo & Duckworth 2000, Pharmacol. Rev. 52, 1-9),an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, aCH4 domain of an immunoglobulin, an albumin binding peptide, or analbumin binding protein, transferrin to name only a few. The albuminbinding protein may be a bacterial albumin binding protein, an antibody,an antibody fragment including domain antibodies (see U.S. Pat. No.6,696,245, for example), or a mutein with binding activity for albumin.Accordingly, suitable conjugation partners for extending the half-lifeof a mutein of the disclosure include an albumin binding protein, forexample, a bacterial albumin binding domain, such as the one ofstreptococcal protein G (König, T., & Skerra, A. (1998) J. Immunol.Methods 218, 73-83). Other examples of albumin binding peptides that canbe used as conjugation partner are, for instance, those having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in U.S. Patent Application 2003/0069395 (incorporatedherein by reference in its entirety) or Dennis et al. (Dennis, M. S.,Zhang, M., Meng, Y. G., Kadkhodayan, M., Kirchhofer, D., Combs, D. &Damico, L. A. (2002) J Biol Chem 277, 35035-35043).

In other embodiments, albumin itself (Osborn, B. L. et al., 2002, J.Pharmacol. Exp. Ther. 303, 540-548), or a biological active fragment ofalbumin can be used as conjugation partner of a mutein of thedisclosure. The term “albumin” includes all mammal albumins such ashuman serum albumin or bovine serum albumin or rat albumine. The albuminor fragment thereof can be recombinantly produced as described in U.S.Pat. No. 5,728,553 or European Patent Applications EP 0 330 451 and EP 0361 991 (incorporated herein by reference in their entirety).Recombinant human albumin (Recombumin®) Novozymes Delta Ltd.(Nottingham, UK) can be conjugated or fused to a mutein of thedisclosure in order to extend the half-life of the mutein.

If the albumin-binding protein is an antibody fragment it may be adomain antibody. Domain Antibodies (dAbs) are engineered to allowprecise control over biophysical properties and in vivo half-life tocreate the optimal safety and efficacy product profile. DomainAntibodies are for example commercially available from Domantis Ltd.(Cambridge, UK and MA, USA).

Using transferrin as a moiety to extend the serum half-life of themuteins of the disclosure, the muteins can be genetically fused to the Nor C terminus, or both, of non-glycosylated transferrin.Non-glycosylated transferrin has a half-life of 14-17 days, and atransferrin fusion protein will similarly have an extended half-life.The transferrin carrier also provides high bioavailability,biodistribution and circulating stability. This technology iscommercially available from BioRexis (BioRexis PharmaceuticalCorporation, PA, USA). Recombinant human transferrin (DeltaFerrin™) foruse as a protein stabilizer/half-life extension partner is alsocommercially available from Novozymes Delta Ltd. (Nottingham, UK).

If an Fc part of an immunoglobulin is used for the purpose to prolongthe serum half-life of the muteins of the disclosure, the SynFusion™technology, commercially available from Syntonix Pharmaceuticals, Inc(MA, USA), may be used. The use of this Fc-fusion technology allows thecreation of longer-acting biopharmaceuticals and may for example consistof two copies of the mutein linked to the Fc region of an antibody toimprove pharmacokinetics, solubility, and production efficiency.

Yet another alternative to prolong the half-life of the muteins of thedisclosure is to fuse to the N- or C-terminus of the muteins long,unstructured, flexible glycine-rich sequences (for example poly-glycinewith about 20 to 80 consecutive glycine residues). This approachdisclosed in WO2007/038619, for example, has also been term “rPEG”(recombinant PEG).

If polyalkylene glycol is used as conjugation partner, the polyalkyleneglycol can be substituted, unsubstituted, linear or branched. It canalso be an activated polyalkylene derivative. Examples of suitablecompounds are polyethylene glycol (PEG) molecules as described in WO99/64016, in U.S. Pat. No. 6,177,074 or in U.S. Pat. No. 6,403,564 inrelation to interferon, or as described for other proteins such asPEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) orPEG-superoxide dismutase (see for example, Fuertges et al. (1990) TheClinical Efficacy of Poly(Ethylene Glycol)-Modified Proteins J. Control.Release 11, 139-148). The molecular weight of such a polymer, such aspolyethylene glycol, may range from about 300 to about 70.000 Dalton,including, for example, polyethylene glycol with a molecular weight ofabout 10.000, of about 20.000, of about 30.000 or of about 40.000Dalton. Moreover, as e.g. described in U.S. Pat. No. 6,500,930 or6,620,413, carbohydrate oligo- and polymers such as starch orhydroxyethyl starch (HES) can be conjugated to a mutein of thedisclosure for the purpose of serum half-life extension.

In addition, a mutein disclosed herein may be fused to a moiety mayconfer new characteristics to the muteins of the disclosure such asenzymatic activity or binding affinity for other molecules. Examples ofsuitable fusion partners are alkaline phosphatase, horseradishperoxidase, gluthation-S-transferase, the albumin-binding domain ofprotein G, protein A, antibody fragments, oligomerization domains ortoxins.

In particular, it may be possible to fuse a mutein disclosed herein witha separate enzyme active site such that both “components” of theresulting fusion protein together act on a given therapeutic target. Thebinding domain of the mutein attaches to the disease-causing target,allowing the enzyme domain to abolish the biological function of thetarget.

The present disclosure also relates to nucleic acid molecules (DNA andRNA) that include nucleotide sequences encoding the muteins of thedisclosure. Since the degeneracy of the genetic code permitssubstitutions of certain codons by other codons specifying the sameamino acid, the disclosure is not limited to a specific nucleic acidmolecule encoding a mutein as described herein but encompasses allnucleic acid molecules that include nucleotide sequences encoding afunctional mutein. In this regard, the present disclosure providesnucleotide sequences, as shown in SEQ ID NOs: 21-34, encoding somemuteins of the disclosure.

In one embodiment of the disclosure, the method includes subjecting thenucleic acid molecule to mutagenesis at nucleotide triplets coding forat least one, or even more, of the sequence positions corresponding tothe sequence positions 28, 36, 40, 41, 49, 52, 65, 68, 70, 72-74, 77,79, 81, 87, 96, 100, 103, 106, 116, 125, 126, 127, 129, 132 and 134 ofthe linear polypeptide sequence of human NGAL (SEQ ID NO: 16).

The disclosure also includes nucleic acid molecules encoding the muteinsof the disclosure, which include additional mutations outside theindicated sequence positions of experimental mutagenesis. Such mutationsare often tolerated or can even prove to be advantageous, for example ifthey contribute to an improved folding efficiency, serum stability,thermal stability or ligand binding affinity of the muteins.

A nucleic acid molecule disclosed in this application may be “operablylinked” to a regulatory sequence (or regulatory sequences) to allowexpression of this nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it includes sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions include a promoter which, inprokaryotes, contains both the promoter per se, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgamo element in prokaryotes or theTATA box, CAAT sequences, and 5′-capping elements in eukaryotes. Theseregions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the disclosure can include aregulatory sequence, such as a promoter sequence. In some embodiments anucleic acid molecule of the disclosure includes a promoter sequence anda transcriptional termination sequence. Suitable prokaryotic promotersare, for example, the tet promoter, the lacUV5 promoter or the T7promoter. Examples of promoters useful for expression in eukaryoticcells are the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the disclosure can also be part of avector or any other kind of cloning vehicle, such as a plasmid, aphagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.

In one embodiment, the nucleic acid molecule is included in a phasmid. Aphasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or f1, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage (e.g.M13K07, VCS-M13 or R408) intact phage particles are produced, therebyenabling physical coupling of the encoded heterologous cDNA to itscorresponding polypeptide displayed on the phage surface (see e.g.Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, orRodi, D. J., and Makowski, L. (1999) Curr. Opin. Blotechnol. 10, 87-93).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a mutein asdescribed herein, replication and control sequences derived from aspecies compatible with the host cell that is used for expression aswell as selection markers conferring a selectable phenotype ontransformed or transfected cells. Large numbers of suitable cloningvectors are known in the art, and are commercially available.

The DNA molecule encoding a mutein as described herein, and inparticular a cloning vector containing the coding sequence of such amutein can be transformed into a host cell capable of expressing thegene. Transformation can be performed using standard techniques. Thus,the disclosure is also directed to a host cell containing a nucleic acidmolecule as disclosed herein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a fusion protein of thedisclosure. Suitable host cells can be prokaryotic, such as Escherichiacoli (E. coli) or Bacillus subtilis, or eukaryotic, such asSaccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells,immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) orprimary mammalian cells.

The disclosure also relates to a method for the production of a muteinas described herein, wherein the mutein or polypeptide, a fragment ofthe mutein or a fusion protein of the mutein is produced starting fromthe nucleic acid coding for the mutein or polypeptide by means ofgenetic engineering methods. The method can be carried out in vivo, themutein or polypeptide can for example be produced in a bacterial oreucaryotic host organism and then isolated from this host organism orits culture. It is also possible to produce a protein in vitro, forexample by use of an in vitro translation system.

When producing the mutein in vivo a nucleic acid encoding such mutein orpolypeptide is introduced into a suitable bacterial or eukaryotic hostorganism by means of recombinant DNA technology (as already outlinedabove). For this purpose, the host cell is first transformed with acloning vector that includes a nucleic acid molecule encoding a muteinas described herein using established standard methods. The host cell isthen cultured under conditions, which allow expression of theheterologous DNA and thus the synthesis of the correspondingpolypeptide. Subsequently, the polypeptide is recovered either from thecell or from the cultivation medium.

In some embodiments, a nucleic acid molecule, such as DNA, disclosed inthis application may be “operably linked” to another nucleic acidmolecule of the disclosure to allow expression of a fusion protein ofthe disclosure. In this regard, an operable linkage is a linkage inwhich the sequence elements of the first nucleic acid molecule and thesequence elements of the second nucleic acid molecule are connected in away that enables expression of the fusion protein as a singlepolypeptide.

In addition, in some embodiments, the naturally occurring disulfide bondbetween Cys 76 and Cys 175 may be removed in hNGAL muteins of thedisclosure. Accordingly, such muteins can be produced in a cellcompartment having a reducing redox milieu, for example, in thecytoplasma of Gram-negative bacteria.

In case a mutein of the disclosure includes intramolecular disulfidebonds, it may be preferred to direct the nascent polypeptide to a cellcompartment having an oxidizing redox milieu using an appropriate signalsequence. Such an oxidizing environment may be provided by the periplasmof Gram-negative bacteria such as E. coli, in the extracellular milieuof Gram-positive bacteria or in the lumen of the endoplasmatic reticulumof eukaryotic cells and usually favors the formation of structuraldisulfide bonds.

It is, however, also possible to produce a mutein of the disclosure inthe cytosol of a host cell, preferably E. coli. In this case, the muteinor polypeptide can either be directly obtained in a soluble and foldedstate or recovered in form of inclusion bodies, followed by renaturationin vitro. A further option is the use of specific host strains having anoxidizing intracellular milieu, which may thus allow the formation ofdisulfide bonds in the cytosol (Venturi et al. (2002) J. Mol. Biol. 315,1-8.).

However, the mutein or polypeptide as described herein may notnecessarily be generated or produced only by use of genetic engineering.Rather, such mutein or polypeptide can also be obtained by chemicalsynthesis such as Merrifield solid phase polypeptide synthesis or by invitro transcription and translation. It is for example possible thatpromising mutations are identified using molecular modeling and then tosynthesize the wanted (designed) polypeptide in vitro and investigatethe binding activity for Ang-2. Methods for the solid phase and/orsolution phase synthesis of proteins are well known in the art (see e.g.Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).

In another embodiment, the mutein or polypeptide of the disclosure maybe produced by in vitro transcription/translation employingwell-established methods known to those skilled in the art.

The skilled worker will appreciate methods useful to prepare muteins orpolypeptides thereof contemplated by the present disclosure but whoseprotein or nucleic acid sequences are not explicity disclosed herein. Asan overview, such modifications of the amino acid sequence include,e.g., directed mutagenesis of single amino acid positions in order tosimplify sub-cloning of a mutated hNGAL gene or its parts byincorporating cleavage sites for certain restriction enzymes. Inaddition, these mutations can also be incorporated to further improvethe affinity of a mutein for its target (e.g. Ang-2 or Ang-1,respectively). Furthermore, mutations can be introduced to modulatecertain characteristics of the mutein such as to improve foldingstability, serum stability, protein resistance or water solubility or toreduce aggregation tendency, if necessary. For example, naturallyoccurring cysteine residues may be mutated to other amino acids toprevent disulphide bridge formation.

The muteins or polypeptides thereof disclosed herein and theirderivatives can be used in many fields similar to antibodies orfragments thereof. For example, the muteins can be used for labelingwith an enzyme, an antibody, a radioactive substance or any other grouphaving biochemical activity or defined binding characteristics. By doingso, their respective targets or conjugates or fusion proteins thereofcan be detected or brought in contact with them. In addition, muteins orpolypeptides thereof of the disclosure can serve to detect chemicalstructures by means of established analytical methods (e.g., ELISA orWestern Blot) or by microscopy or immunosensorics. In this regard, thedetection signal can either be generated directly by use of a suitablemutein conjugate or fusion protein or indirectly by immunochemicaldetection of the bound mutein via an antibody.

Additional objects, advantages, and features of this disclosure willbecome apparent to those skilled in the art upon examination of thefollowing Examples and the attached Figures thereof, which are notintended to be limiting. Thus, it should be understood that although thepresent disclosure is specifically disclosed by exemplary embodimentsand optional features, modification and variation of the disclosuresembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure.

V. EXAMPLES Example 1: Selection of Muteins Specifically Binding toAng-2

Libraries, generated by random mutagenesis of mature hNGAL, were usedfor selection of muteins specifically binding to the human Ang-2.

2×10¹² phagemids from these libraries were incubated with 200 nMbiotinylated human Ang-2 (R & D System). Paramagnetic beads coated withneutravidin or streptavidin were used to capture target/phagemidcomplexes which were subsequently isolated with a magnet. Unboundphagemids were removed by washing the beads with PBST or PBS. Boundphagemids were first eluted with 300 μl 70 mM triethylamine followed byimmediate neutralization of the supernatant with 100 μl 1M Tris-Cl pH6.0. After one intermediate wash cycle remaining phagemids were elutedwith 100 mM glycin pH 2.2 for 10 min followed by immediateneutralization with 50 μl 0.5 M Tris-base. Both elution fractions werepooled and used to infect 4 ml of E. coli XL1-blue culture forreamplification.

Four consecutive rounds of selection were performed. Phagemid DNA wasprepared from E. coli cells infected with the output of the fourthselection round and an hNGAL mutein cassette was isolated by digestion.The hNGAL mutein cassette was inserted into the likewise cut vector,which allows bacterial production of the hNGAL muteins under the controlof a tetracyclin promoter. CaCl2-competent TG1-F′ cells were transformedwith the ligation mixture and plated on LB/Amp plates.

For optimization of Ang-2 specific muteins, additional libraries weregenerated based on mutein SEQ ID NO: 1 and SEQ ID NO: 3. Libraries weregenerated using either a biased randomization of selected positions orerror prone polymerase chain reaction (PCR) based methods. Selection ofoptimized muteins was performed as described above but with increasedstringency.

Example 2: Identification of Muteins Specifically Binding to Ang-2 UsingHigh-Throughput ELISA Screening

Individual colonies were used to inoculate 2xYT/Amp medium and grownovernight (14-18 h) to stationary phase. Subsequently, 50 μl 2xYT/Ampwere inoculated from the stationary phase cultures and incubated for 3 hat 37° C. and then shifted to 22° C. until an OD % of 0.6-0.8 wasreached. Production of muteins was induced by addition of 10 μl 2xYT/Ampsupplemented with 1.2 μg/ml anhydrotetracyclin. Cultures were incubatedat 22° C. until the next day. After addition of 40 μl of 5% (w/v) BSA inPBS/T and incubation for 1 h at 25° C. cultures were ready for use inscreening assays.

Specific binding of the isolated muteins to human Ang-2 was tested bycoating a 1:1 mixture of neutravidin and streptavidin (5 μg/ml in PBS)overnight at 4° C. on microtiterplates. After blocking the plate 1 hwith 2% BSA in PBST the biotinylated target used for selection wascaptured on the coated microtiterplates at a concentration of 1 μg/ml inPBS/T. Plates coated in the same manner without biotinylated target wereused as negative control target in the screening. Subsequently, 20 μl ofBSA-blocked cultures were added to the coated microtiter platecontaining either captured target or aldosterone and incubated for 1 hat 25° C. Bound muteins were detected after 1 h incubation with ananti-Streptag antibody conjugated with horseradish peroxidase (IBA,Boettingen). For quantification, 20 μl of QuantaBlu fluorogenicperoxidase substrate was added and the fluorescence determined at anexcitation wavelength of 320 nm and an emission wavelength of 430 nm.Muteins specifically binding to Ang-2 were then sequenced.

To select for muteins with increased affinity and stability, screeningwas performed with i) reduced antigen concentration and/or ii)competition with Tie-2 Fc (Reliatech) and/or iii) incubation of thescreening supernatant at 65° C. or 70° C. before addition to the targetplate and/or iv) using reverse screening formats were the muteins werecaptured via the Streptag on microtiter plates coated with anti-Streptagantibody (Qiagen) and different concentrations of target was added anddetected via HRP-labelled anti-His-tag antibody (Abcam).

Example 3: Expression of Muteins

Unique muteins were expressed with C-terminal sequence (SEQ ID NO: 15)including the Strep-tag® II, in 2YT-Amp media to purify the muteinsafter expression using Strep-Tactin affinity chromatography andpreparative size exclusion chromatography were applicable.

Example 4: Affinity of Muteins to Ang-2 in an ELISA Based Setting

Binding of muteins was tested by a sandwich ELISA assay. In detail, a384-well plate suitable for fluorescence measurements (Greiner FLUOTRAC™600, black flat bottom, high-binding) was coated with 20 μl of Ang-2 ata concentration of 5 μg/ml in PBS over night at 4 C. After washing, theAng-2-coated wells were blocked with 100 μl blocking buffer containing0.1% Tween 20 and 2% BSA (PBS-T/BSA) for 1 h at room temperature.

20 μl of serially diluted muteins were incubated in PBS-T/BSA for 1 h atroom temperature (RT).

The residual supernatants were discarded and 20 μl HRP-labeledanti-Streptag antibody was added at a predetermined optimalconcentration in PBS-T/BSA and incubated for 1 h at RT. After washing,20 μl fluorogenic HRP substrate (QuantaBlu, Thermo) was added to eachwell, and the reaction was allowed to proceed for 15 to 60 minutes. Thefluorescence intensity of every well on the plate was read using afluorescence microplate reader (Tecan or Molecular Devices). Curvefitting was performed using GraphPad Prism 4 software. The resultingEC50 values are summarized in Table 1 below.

TABLE 1 EC50 mean Mutein SEQ ID [nM] SEQ ID NO: 1 3.7 SEQ ID NO: 2 3 SEQID NO: 3 2.8 SEQ ID NO: 5 3.5 SEQ ID NO: 7 2.1 SEQ ID NO: 8 2.4 SEQ IDNO: 9 2.1 SEQ ID NO: 11 2.3

Example 5: Competitive Mode of Action of Lipocalin Mutein Binding toAng-2

Whether the selected muteins bind to human Ang-2 in a competitive modewas tested in vitro using a competition ELISA format assay (seeexemplary lipocalin muteins in FIG. 1). In this experiment, a constantconcentration of human Ang-2 was incubated with variable concentrationsof lipocalin muteins for 1 h. After this pre-incubation in solution, analiquot of the lipocalin mutein/l Ang-2 mixture was transferred to anELISA plate coated with human Tie-2 receptor to measure theconcentration of hAng-2 that was not blocked to bind to hTie-2.

All incubation steps were performed with shaking at 300 rpm, and theplate was washed after each incubation step with 80 μl PBS-T buffer(PBS, 0.05% Tween 20) for five times using a Biotek EL405 select CWwasher (Biotek). In the first step, a 384 well plate was directly coatedwith 20 μl of soluble human hTie-2 Fc (Reliatech) at a concentration of2 μg/ml in PBS over night at 4° C. After washing, h Tie-2 Fc coatedwells were blocked with 60 μl PBS-T/BSA (2% BSA in PBS containing 0.1%Tween 20) for 1 h at room temperature.

A fixed concentration of 0.5 nM human Ang-2 was incubated in solutionwith varying concentrations of SEQ ID NOs: 1-14, or with SEQ ID NO: 16as a negative control and with benchmark antibodies 1 and 2 (SEQ ID NOs:17/18 and 19/20, respectively) as a positive controls, using a suitablestarting concentration which was serially diluted at a 1:3 ratio down tothe picomolar range in PBS-T/BSA buffer. After 1 h incubation at roomtemperature. 20 μl of the reaction mixture was transferred to the hTie-2Fc-coated plate to capture unbound (free) or non-competitively boundhAng-2 for 20 min at RT. To allow for transformation of ELISA readoutresults into absolute free Ang-2 concentrations (cf. below), a standardcurve containing varying concentrations of hAng-2 was prepared inPBS-T/BSA and incubated for 20 min on the same plate as well.

To allow for detection and quantitation of bound hAng-2, the residualsupernatants were discarded and 20 μl anti-HIS-tag HRP-labeled antibodywas added at a concentration in PBS-T/BSA and incubated for 1 h at RT.After washing, 20 μl quanta blue was added to each well and thefluorescence intensity of every well was read using a fluorescencemicroplate reader (Tecan or Molecular Devices).

The evaluation was performed as follows: free hAng-2 concentrationc(Ang-2)_(free) was calculated (from the standard curve determined inparallel) and plotted versus SEQ ID NOs: 1-14, 16, and benchmarkantibodies 1 and 2 (SEQ ID NOs: 17/18 and 19/20, respectively)concentration, c(SEQ ID NOs: 1-14, 16, 17/18 and 19/20). To obtain theSEQ ID NOs: 1-14, 16, 17/18 and 19/20 concentration at which formationof the hAng-2/hTie-2 Fc-complex was blocked by 50% (IC50), the curveswere fitted by nonlinear regression with a single-sites binding modelaccording to c(Ang-2)_(free)=c(Ang-2)_(tot)/(1+c(SEQ ID NOs: 1-14, 16,17/18 and 19/20)/IC50)), with the total tracer concentrationc(Ang-2)_(tot) and the IC50 value as free parameters (as shown in FIG.1). Curve fitting was performed using GraphPad Prism 4 software. Theresulting IC50 values are summarized in Table 2 below.

TABLE 2 IC50 mean Mutein SEQ ID [nM] SEQ ID NO: 1 0.5581 SEQ ID NO: 21.711 SEQ ID NO: 3 0.3194 SEQ ID NO: 4 0.4123 SEQ ID NO: 5 0.8613 SEQ IDNO: 6 0.06273 SEQ ID NO: 7 0.06193 SEQ ID NO: 8 0.07083 SEQ ID NO: 90.09287 SEQ ID NO: 10 0.07940 SEQ ID NO: 11 0.09877 SEQ ID NO: 12 0.1312SEQ ID NO: 13 0.1842 SEQ ID NO: 14 0.1054 SEQ ID NO: 19/20 0.03067 SEQID NO: 17/18 0.01580

Example 6: Affinity of Muteins Binding to Ang-2 Determined in Biacore

In a Surface Plasmon Resonance (SPR) based assay, a Biacore T200instrument (GE Healthcare) was used to measure the binding affinity ofmuteins to hAng-2. Muteins selected for binding to Ang-2 and negativecontrol (SEQ ID NO: 16) were biotinylated for 2 h at room temperatureapplying an appropriate excess of EZ-Link NHS-PEG4-Biotin (Thermo).Biotinylated samples were purified from non-reacted Biotin using a ZebaSpin Desalting Plate (Thermo) according to the manufacturesinstructions.

In the SPR affinity assay, biotinylated muteins and negative controlwere captured on a sensor chip CAP using the Biotin CAPture Kit (GEHealthcare): Sensor Chip CAP is pre-immobilized with an ssDNA oligo.Undiluted Biotin CAPture Reagent (streptavidin conjugated with thecomplementary ss-DNA oligo) was applied at a flow rate of 2 μl/min for300 s. Subsequently, 0.02 μg/mL of biotinylated muteins or negativecontrol were applied for 300 s at a flow rate of 5 μl/min. As theanalyte Ang-2 is a multimeric molecule, it was aimed to create a surfacewith minimal ligand density to reduce bivalent interactions on the chipsurface to a minimum. The reference channel was loaded with BiotinCAPture Reagent only.

To determine the binding affinity, four dilutions of human Ang-2 atconcentrations in the range of 1-27 nM were prepared in HBS-EP+ buffer(GE Healthcare) and applied to the prepared chip surface. Applying aflow rate of 30 μL/min, a multi cycle kinetics approach was used with asample contact time of 180 s and a dissociation time of 4600 s. Allmeasurements were performed at 25° C. Regeneration of the Sensor ChipCAP surface was achieved with an injection of 6 M Gua-HCl with 0.25 MNaOH followed by an extra wash with running buffer or water and astabilization period of 120 s. Data were evaluated with Biacore T200Evaluation software (V 1.0). Double referencing was used. A 1:1 Bindingmodel was used to fit the raw data even though the analyte was known tobe a multimer.

The resulting kinetic constants for a selection of lipocalin muteins aresummarized in Table 3 below. Such exemplary lipocalin muteins (SEQ IDNOs: 7, 8, 9 and 11) bind human Ang-2 with affinities in the range of0.2-1.4 nM while no binding is detectable for the negative control (SEQID NO: 16). It has to be noted, that the fitted values were derivedbased on the assumption that using a very low ligand density with Rmaxvalues below ten reduces multivalent interactions to a negligible level.

TABLE 3 k_(on) k_(off) KD Rmax Mutein SEQ ID [M⁻¹*s¹] [s⁻¹] [nM] [RU]SEQ ID NO: 9 3.2E+05 9.4E−05 0.29 5.4 SEQ ID NO: 7 1.8E+05 2.6E−04 1.438.1 SEQ ID NO: 8 3.5E+05 1.3E−04 0.37 6.5 SEQ ID NO: 11 4.0E+06 8.7E−040.21 2.6

Example 7: Specificity and Species Crossreactivity of Lipocalin Muteinto Ang-1 and Ang-2

Specificity and species crossreactivity of the lipocalin muteins (SEQ IDNOs: 1, 3, 7, 8, 9, 11) and of the benchmark antibody (SEQ ID NOs:17/18) were tested by a “solution competition ELISA” assay (seeexemplary lipocalin muteins FIG. 2), the principle of which was asfollows: A constant concentration of SEQ ID NOs: 1, 3, 7, 8, 9, 11, andbenchmark antibody (SEQ ID NOs: 17/18) was incubated with variableconcentrations of ligands (human Ang-2, human Ang-1, human Ang-4, mouseAng2 and mouse Ang 3 as well as hVEGF-A as negative control (R&Dsystem)) or 1 h. After this pre-incubation in solution, an aliquot ofthe mutein/ligand mixture was transferred to an ELISA plate with hAng-2immobilized to measure the remaining concentration of free lipocalinmuteins SEQ ID NOs: 1, 3, 7, 8, 9, 11, and of free benchmark antibody.The concentration of free (unbound) SEQ ID NOs: 1, 3, 7, 8, 9, 11, andof free benchmark antibody was determined via a quantitative ELISAsetup. Note that this assay relied on that all ligands were targetingthe same binding site on SEQ ID NOs: 1, 3, 7, 8, 9, 11, and 17/18, i.e.that the ligands bound to the SEQ ID NOs: 1, 3, 7, 8, 9, 11, and 17/18in competition with each other.

In the following detailed experimental protocol, incubation and washingsteps were performed as described above in the competition ELISAprotocol (see Example 5). A 384-well plate suitable for fluorescencemeasurements (Greiner FLUOTRAC™ 600, black flat bottom, high-binding)was coated with 20 μl of h Angiopoietin 2 of 5 μg/ml in PBS over nightat 4° C. After washing, the Ang-2-coated wells were blocked with 100 μlblocking buffer (PBS-T/BSA) for 1 h at room temperature.

A fixed concentration of 0.1 nM of SEQ ID NOs: 1, 3, 7, 8, 9, 11, and17/18 were incubated in solution with varying concentrations of ligands(hAng-2, mAng-2, hAn1, mAng-3 and hAng-4 and VEGF-A), using a suitablestarting concentration which was serially diluted at a 1:3 ratio down tothe picomolar range in PBS-T/BSA. After 1 h incubation at roomtemperature, 20 μl of the reaction mixture was transferred to the384-well plate upon which hAng-2 was immobilized to capture unbound(free SEQ ID NOs: 1, 3, 7, 8, 9, 11, and 17/18 for 20 min at RT. Toallow for transformation of ELISA readout results into absolute free SEQID NOs: 1, 3, 7, 8, 9, 11, and 17/18 concentrations (cf. below), astandard curve containing varying concentrations SEQ ID NOs: 1, 3, 7, 8,9, 11, and 17/18 were prepared in PBS-T/BSA and incubated for 20 min onthe same ELISA plate as well.

The residual supernatants were discarded and 20 μl HRP-labeledanti-lipocalin-mutein antibody was added at a predetermined optimalconcentration in PBS-T/BSA and incubated for 1 h at RT. Theanti-lipocalin-mutein antibody had been obtained by immunization ofrabbits with a mixture of lipocalin muteins, and was subsequentlycoupled to HRP using a kit (EZ-link Plus Activated Peroxidase, ThermoScientific) according to the manufacturer's instructions, to obtain theantibody-HRP conjugate. After washing, 20 μl fluorogenic HRP substrate(Quantablue, Pierce) was added to each well, and the reaction wasallowed to proceed for 60 minutes. The fluorescence intensity of everywell on the plate was read using a Genios Plus Microplate reader(Tecan). To evaluate the data, free muteins concentration,c(Mutein)_(free), was calculated based on the standard curve results,and plotted versus ligand concentration, c(Ligand). To obtain the ligandconcentration at which formation of the hAng-2/Mutein complex wasblocked by 50% (IC50), the curves were fitted by nonlinear regressionwith a single-sites binding model according toc(Mutein)_(free)=c(Mutein)_(tot)/(1+c(Ligand)/IC50)), with the totaltracer concentration c(Mutein)_(tot) and the IC50 value as freeparameters. Curve fitting was performed using GraphPad Prism 4 software.Said curve fitting yielded the results that are summarized in thefollowing Table 4.

TABLE 4 Solution bindin IC58: nM g ELISA hAng-1 hAng-2 mAng-2 hAng-4mAng-3 hVEGF-A SEQ ID NO: 17/18 26 0.6 0.07 N/A N/A N/A SEQ ID NO: 3 N/A1.4 1.8 N/A N/A N/A SEQ ID NO: 1 >100 2.5 0.65 N/A N/A N/A SEQ ID NO: 110.89 0.49 0.05 N/A N/A N/A SEQ ID NO: 8 9.3 0.5 0.08 N/A N/A N/A SEQ IDNO: 7 0.47 0.41 0.05 N/A N/A N/A SEQ ID NO: 9 1.9 0.76 0.11 N/A N/A N/A

As depicted in FIG. 2, the data demonstrates that SEQ ID NO: 8 displayedhigh affinity towards human and mouse Ang-2 and SEQ ID NO: 7 displayedhigh affinity not only towards human and mouse Ang-2 but also towardshuman Ang-1.

Example 8: Lipocalin Muteins Blocking the Binding of Human and MouseAng-2 to hTie-2 Expressing Cells

Whether lipocalin muteins binds to human Ang-2 and mouse Ang-2 in acompetitive mode was tested on hTie-2 overexpressing HEK cells using acompetition cell electrochemoluminescence (ECL) assay format (see FIGS.3 and 4). In this experiment, a constant concentration of human Ang-2and mouse Ang-2 was incubated with variable concentrations of lipocalinmuteins (SEQ ID NOs: 1, 3, 6-14) and benchmark antibody (SEQ ID NOs:19/20) for 1 h. After this pre-incubation in solution, an aliquot of thelipocalin mutein/Ang-2 mixture was transferred to an MSD plate coatedwith hTie-2 overexpressing HEK cells to measure the concentration ofhAng-2 or mAng-2 that was not blocked to bind to hTie-2 respectively.

All incubation steps were performed at room temperature, and the platewas washed after each incubation step with 80 μl PBS buffer for twotimes using a Biotek EL405 select CW washer (Biotek). In the first step,a 384 well plate was precoated for 5 minutes with poly D lysine andwashed twice with PBS. 10⁴ HEK:hTie-2 cells per well were seeded andallowed to adhere to the surface of the wells overnight at 37° C. Afterwashing, cell coated wells were blocked with 60 μl PBS/Casein (2% Caseinin PBS) for 1 h at room temperature.

A fixed concentration of human Ang-2 or of mouse Ang-2 was incubated insolution with varying concentrations of SEQ ID NOs: 1, 3, 6-14 and ofbenchmark antibody (SEQ ID NO: 19/20) using a suitable startingconcentration of SEQ ID NOs: 1, 3, 6-14 and of benchmark antibody whichwas serially diluted at a 1:3 ratio down to the picomolar range inPBS-/Casein buffer. After 1 h incubation at room temperature, 20 μl ofthe reaction mixture was transferred to the HEK:hTie2-coated plate tocapture competitively unbound hAng-2 for 1 hour min at RT. A standardcurve containing varying concentrations of hAng-2 or mouse Ang-2 wasprepared in PBS/Casein and incubated for 1 hour the same plate as well.

To allow for detection and quantitation of bound hAng-2 and mouse Ang-2,the residual supernatants were discarded and 20 μl of a mixtureanti-HIS-tag antibody (Abcam) and Sulfotag labelled anti-goat antibody(Mesoscale Discovery) was added at a concentration of 1 μg/ml inPBS/casein and incubated for 1 h at RT. After washing, 35 μlsurfactant-free reading buffer was added to each well and the ECL signalof every well was read using a Mesoscale Discovery reader.

Evaluation and curve fitting was performed using GraphPad Prism 4software and the yielded results are summarized in Table 5 below.

TABLE 5 hAng2 IC50 mAng-2 IC50 mutein SEQ ID [nM] [nM] SEQ ID NO: 310.73 23.17 SEQ ID NO: 1 18.91 3.982 SEQ ID NO: 6 3.464 1.206 SEQ ID NO:7 3.596 1.228 SEQ ID NO: 8 3.746 1.245 SEQ ID NO: 9 3.817 1.39 SEQ IDNO: 10 4.626 1.046 SEQ ID NO: 11 4.793 1.448 SEQ ID NO: 12 5.287 3.118SEQ ID NO: 13 5.431 7.343 SEQ ID NO: 14 6.41 2.32 SEQ ID NO: 19/20 1.9990.8826

Example 9: Lipocalin-Mutein-Mediated Blockade of Ang-2 in Cell-BasedProliferation Assay

The ability of the lipocalin muteins of SEQ ID NOs: 1, 3, 7-9 and 11 toneutralize the biological activity of hAng-2 was assessed by theapplication of a short-term proliferation bioassay employing lymphaticmicrovascular endothelial cells (LEC). The LEC proliferation can beinhibited by agents having hAng-2-neutralizing effect. In the assay, SEQID NOs: 1, 3, 7-9 and 11 were added to serum starved LEC cells inculture. After three days in culture, the extent of proliferation wasassessed by quantifying the number of viable cells. This was performedusing the CellTiter-Glo Luminescent Cell Viability Assay (Promega) tomeasure ATP levels, which correlate with the number of metabolicallyactive cells. The ability of SEQ ID NOs: 1, 3, 7-9 and 11 to neutralizehAng-2 was assessed by their IC50 value. i.e. the concentration of thelipocalin muteins that lead to half-maximal inhibition of hAng-2mediated proliferation.

The detailed procedure of setting up the assay is hereby described inthe following. LEC were maintained in EBM, 5% fetal calf serum and MV2supplemental kit. Assays were performed in 96 wells white clearflat-bottom plates (Greiner) in 25 μL per well.

LEC cells were cultured in cell culture flasks under standard conditionsaccording to manufacturer's instruction (PAA Laboratories), 37° C., 5%CO₂ atmosphere).

On day 1 of the experiment, the adherent cells were dissociated fromtheir substrate with trypsine/EDTA according to the manufacturer'sinstructions. Subsequently, cells were centrifuged down for 5 minutes at1000 rpm, resuspended in EBM and filtered through a 100 μm cell strainer(Falcon) to remove cell aggregates. Cells were then seeded in 96-wellflat bottom tissue culture plates (Greiner) at a density of 3200 cellsper well using an end volume of 100 μl. They were incubated 1 hour understandard conditions.

After 1 h dilution series of SEQ ID NOs: 1, 3, 7-9, 11, of negativecontrols (SEQ ID NO: 16 and a human IgG isotype antibody (Dianova)) andof benchmark antibodies 1 and 2 (SEQ ID NOs: 17/18 and 19/20,respectively) were added to the wells. All titration series wereperformed with a serial 1:3 dilution in assay medium and a suitablestarting concentration. Subsequently, the cells were allowed toproliferate for 72 hours at 37° C. To quantify cell proliferation after72 hours, 25 μL CellTiter-Glo reagents were added to the cells in eachof the wells and incubated for 2 minutes on an orbital shaker to inducecell lysis, and luminescence was measured using the PheraStar FS reader.

IC50 values were determined using GraphPad Prism software (GraphPadSoftware Inc.) by plotting standardized signal agains samples'concentration and non-linear regression of the data with a sigmoidaldose-response model.

The result of the experiment is shown in FIG. 5. The proliferation assaydisclosed above is representative of three independent experiments. Theresults are summarized in Table 6 below.

TABLE 6 IC50 mean Mutein SEQ ID [nM] SEQ ID NO: 1 2.252 SEQ ID NO: 32.806 SEQ ID NO: 7 0.1992 SEQ ID NO: 8 0.0780 SEQ ID NO: 9 0.1423 SEQ IDNO: 11 0.1692 SEQ ID NO: 17/18 0.0668 SEQ ID NO: 19/20 0.0435

SEQ ID NO: 8 displayed an average EC50 of 0.07 nM, benchmark antibody 1(SEQ ID NOs: 17/18) exhibited an EC50 of 0.06 nM, and benchmark antibody2 (SEQ ID NOs: 19/20) exhibited an EC50 of 0.04 nM. The negativecontrols had no effect on proliferation. The data therefore demonstratesthat SEQ ID NO: 8 and the benchmark antibodies exhibit a comparablepotency in this functional assay.

Example 10: Stability Assessment of Muteins

The resulting melting temperatures (Tm) as well as the onset of meltingfor the lipocalin muteins (SEQ ID NOs: 1, 3, 7-9, 11) are listed inTable 7 below in comparison with benchmark antibody (SEQ ID NOs: 17/18).The lipocalin muteins selected have Tm ranging from 65 to 77° C.,indicating good stability of the molecules.

TABLE 7 Tm and onset of melting as determined by nanoDSC of Ang-2specific lipacalin muteins and benchmark antibody Tm onset of meltingMutein SEQ ID [° C.] [°C.] SEQ ID NO: 1 64 59 SEQ ID NO: 3 66 59 SEQ IDNO: 7 76 68 SEQ ID NO: 8 73 63 SEQ ID NO: 9 77 70 SEQ ID NO: 11 75 65SEQ ID NO: 17/18 69.5/76.1/83.4 64

To assess storage stability, exemplary muteins at a conc. of 1 mg/ml inPBS were incubated for 1 week at 37° C. Active mutein was measured in aquantitative ELISA setting. Monomeric protein was measured in ananalytical size exclusion chromatography. Resulted data for SEQ ID NOs:1, 3, 7-9 and 11 are shown in Table 8 below.

To test protein activity, the following ELISA was applied: A 384-wellplate suitable for fluorescence measurements (Greiner FLUOTRAC™ 600,black flat bottom, high-binding) was coated with 20 μL of Ang-2 (ThermoScientific) at a concentration of 5 μg/ml in PBS overnight at 4° C.After washing, the Ang-2-coated wells were blocked with 100 μl blockingbuffer (2% w/v BSA in PBS containing 0.1% v/v Tween-20) for 1 h. Theplate was washed and 20 μl of appropriately diluted protein standard,unstressed reference sample or stressed sample was transferred to theELISA plate and incubated. To quantitate plate-bound protein, the ELISAplate was washed, residual supernatants were discarded and 20 μlHRP-labeled anti-hNGAL antibody was added at a predetermined optimalconcentration in blocking buffer and incubated. After washing, 20 μlfluorogenic HRP substrate (QuantaBlu, Pierce) was added to each well,and the reaction was allowed to proceed for 20-30 minutes. Thefluorescence intensity of every well on the plate was read using afluorescence microplate reader (Tecan).

Unless otherwise stated all incubation steps were performed at for 1 hat room temperature and after each incubation step the plate was washedwith 100 μl PBS-T buffer (PBS, 0.05% Tween 20) for five times using aBiotek ELx405 select CW washer.

For the ELISA described above, a calibration curve including 11dilutions typically ranging from 0.008-500 ng/mL was prepared and threedifferent, independent dilutions within the linear range of thecalibration curve were prepared for each sample. Blocking bufferoptionally supplemented with 1% human or murine plasma was used for thedilutions.

The calibration curve was fit using a 4 Parameter Logistic (4PL)nonlinear regression model and used to calculate active proteinconcentrations for the tested samples. The determined active proteinconcentrations were referenced against an unstressed sample stored atthe same concentration and in the same matrix.

Analytical size exclusion chromatography was performed on an AgilentHPLC system with two Superdex 75 5/150 GL columns (GE Healthcare) in arow using PBS (Gibco) as an eluent at a flow rate of 0.3 mL/min.

To assess storage stability in plasma, exemplary muteins at a conc. of0.5 mg/ml were incubated for 1 week at 37° C. in human and mouse plasma.Active mutein was measured in a quantitative ELISA setting as describedabove.

All tested lipocalin muteins proved to be stable under all testedconditions and the test results are summarized in Table 8 below.

TABLE 8 Stability after 3 freeze/thaw cycles (F/T); 1 week storage inPBS at 37° C. and 1 week storage in human (hu) or mouse (mu) plasmaassessed by recovery of activity in qELISA and monomer content inanalytical SEC: stable in qELISA = 100 +/− 15%; stable in aSEC = 100 +/−5% (recovery of monomer peak area compared to non-stressed referencesample); for all samples including references a monomer content of 100area percent has been detected. 1 week hu 1 week mu Freeze/thaw 1 weekPBS37° C. plasma Plasma Mutein SEQ ID Q EliSA HPLC Q EliSA HPLC Q EliSAQ EliSA SEQ ID NO: 2 104 100 94 97 — — SEQ ID NO: 3 95 98 100 96 — — SEQID NO: 1 102 103 99 105 — — SEQ ID NO: 7 — — — — 99 85 SEQ ID NO: 8 — —— — 89 93 SEQ ID NO: 9 — — — — 99 100 SEQ ID NO: 11 — — — — 100 89 SEQID NO: 17/18 108 109 87 103 — —

Embodiments illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present embodiments have been specificallydisclosed by preferred embodiments and optional features, modificationand variations thereof may be resorted to by those skilled in the art,and that such modifications and variations are considered to be withinthe scope of this invention. All patents, patent applications, textbooksand peer-reviewed publications described herein are hereby incorporatedby reference in their entirety. Furthermore, where a definition or useof a term in a reference, which is incorporated by reference herein isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosurealso forms part of the invention. This includes the generic descriptionof the invention with a proviso or negative limitation removing anysubject matter from the genus, regardless of whether or not the excisedmaterial is specifically recited herein. In addition, where features aredescribed in terms of Markush groups, those skilled in the art willrecognize that the disclosure is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Furtherembodiments will become apparent from the following claims.

1-58. (canceled)
 59. A nucleic acid molecule comprising a nucleotidesequence encoding a human neutrophil gelatinase-associated lipocalin(hNGAL) mutein capable of binding angiopoietin-2 (Ang-2) with detectableaffinity, wherein the mutein has a sequence identity of at least 85% toa sequence selected from the group consisting of SEQ ID NOs: 1-14. 60.The nucleic acid molecule of claim 59, wherein the mutein comprises thefollowing mutated amino acid residue in comparison with the linearpolypeptide sequence of mature hNGAL (SEQ ID NO: 16): Gln 28→His. 61.The nucleic acid molecule of claim 59, wherein the mutein comprises atleast fifteen of the following mutated amino acid residues in comparisonwith the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 16):Gln 28→His; Leu 36→Gln, Glu, His, Val, Met or Phe; Ala 40→Val, Tyr, Hisor Trp; Ile 41→His, Tyr, Trp, Val, Arg, Glu, or Asp; Gln 49→Gly, Ile,Glu or Val; Tyr 52→Trp, His, Thr or Ser; Ser 68→Gly, Asp, Gln, Glu orIle; Leu 70→Ser, Thr, Gly, Arg, Tyr or Ala; Arg 72→Gly, Ala, Trp, Thr orGlu; Lys 73→Pro, Phe, Leu, Arg, Ala or Gln; Asp 77→Asn, Lys, Ser or Val;Trp 79→Thr, Arg, Ser or Asn; Arg 81→Trp, His or Tyr; Asn 96→Gly, Ala,Pro, Gln or Asp; Tyr 100→Pro, Trp, Gly, Ser, Leu or Asp; Leu 103→Gly,Glu, Asp, Met or Gln; Tyr 106→Thr, Leu, Phe, or Pro; Lys 125→His, Thr orGly; Ser 127→Leu, Met, or Tyr; Tyr 132→Phe, Trp or Val; and Lys 134→Ala,Glu or Trp.
 62. The nucleic acid molecule of claim 59, wherein themutein is capable of binding Ang-2 with a dissociation constant (KD) ofabout 5 nM or lower.
 63. The nucleic acid molecule of claim 59, whereinthe mutein is capable of binding Ang-2 with an affinity measured by anEC50 value of about 5 nM or lower.
 64. The nucleic acid molecule ofclaim 59, wherein the mutein is capable of binding Ang-2 with anaffinity measured by an IC50 value of about 5 nM or lower.
 65. Thenucleic acid molecule of claim 59, wherein the mutein is capable ofinhibiting or reducing lymphatic microvascular endothelial cellproliferation mediated by Ang-2 with an IC50 value of about 5 nM orlower.
 66. The nucleic acid molecule of claim 59, wherein the muteincomprises one or more of the following mutated amino acid residues incomparison with the linear polypeptide sequence of mature hNGAL (SEQ IDNO: 16): Asn 65→Asp; Lys 74→Glu; Cys 87→Ser; Asn 116→Asp; Val 126→Met;and Asn 129→Asp.
 67. The nucleic acid molecule of claim 59, wherein themutein comprises one of the following sets of mutated amino acidresidues in comparison with the linear polypeptide sequence of maturehNGAL (SEQ ID NO: 16): (a) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Gln49→Gly; Tyr 52→Trp; Ser 68→Gly; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp77→Asn; Trp 79→Thr; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr 100→Pro; Leu103→Gly; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr 132→Phe; Lys134→Glu; (b) Gln 28→His; Leu 36→Phe; Ala 40→His; Ile 41→Arg; Gln 49→Gly;Tyr 52→His; Ser 68→Asp; Leu 70→Thr; Arg 72→Ala; Lys 73→Phe; Asp 77→Asn;Trp 79→Arg; Arg 81→His; Cys 87→Ser; Tyr 100→Trp; Leu 103→Glu; Tyr106→Thr; Lys 125→Thr; Ser 127→Met; Tyr 132→Trp; Lys 134→Trp; (c) Gln28→His; Leu 36→Val; Ala 40→Trp; Ile 41→Tyr; Gln 49→Ile; Tyr 52→Thr; Ser68→Gln; Leu 70→Gly; Arg 72→Glu; Lys 73→Gln; Asp 77→Lys; Trp 79→Ser; Arg81→His; Cys 87→Ser; Asn 96→Asp; Tyr 100→Trp; Leu 103→Asp; Tyr 106→Leu;Lys 125→Gly; Ser 127→Met; Tyr 132→Val; Lys 134→Ala; (d) Gln 28→His; Leu36→Glu, Ala 40→Val; Ile 41→Glu; Gln 49→Val; Tyr 52→Thr Ser 68→Glu; Leu70→Arg; Arg 72→Trp; Lys 73→Leu; Asp 77→Lys; Trp 79→Asn; Arg 81→His; Cys87→Ser; Asn 96→Ala; Tyr 100→Gly; Leu 103→Met; Tyr 106→Thr; Lys 125→Thr;Ser 127→Met; Tyr 132→Trp; Lys 134→Trp; (e) Gln 28→His; Leu 36→Gln; Ala40→Tyr; Ile 41→Trp; Gln 49→Ile; Tyr 52→Ser; Ser 68→Ile; Leu 70→Tyr; Arg72→Thr; Lys 73→Arg; Asp 77→Ser; Trp 79→Arg; Arg 81→Tyr; Cys 87→Ser; Asn96→Pro; Leu 103→Asp; Tyr 106→Thr; Lys 125→His; Ser 127→Tyr; Tyr 132→Trp;Lys 134→Glu; (f) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Gln 49→Glu; Tyr52→Trp; Asn 65→Asp; Ser 68→Gly; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp77→Asn; Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr 100→Ser; Leu103→Gln; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr 132→Phe; Lys134→Glu; (g) Gln 28→His; Leu 36→His; Ala 40→Tyr; Gln 49→Glu; Tyr 52→Trp;Asn 65→Asp; Ser 68→Glu; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp 77→Asn;Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr 100→Pro; Leu103→Asp; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr 132→Phe; Lys134→Glu; (h) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Gln 49→Gly; Tyr 52→Trp;Asn 65→Asp; Ser 68→Glu; Leu 70→Ser; Arg 72→Gly; Lys 73→Ala; Asp 77→Asn;Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr 100→Asp; Leu103→Gly; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr 132→Phe; Lys134→Glu; (i) Gln 28→His; Leu 36→His; Ala 40→Tyr; Gln 49→Gly; Tyr 52→Trp;Asn 65→Asp; Ser 68→Glu; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp 77→Asn;Trp 79→Arg; Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr 100→Pro; Leu103→Gly; Tyr 106→Thr; Lys 125→His; Ser 127→Leu; Tyr 132→Phe; Lys134→Glu; (j) Gln 28→His; Leu 36→Gln; Ala 40→Tyr; Tyr 52→Trp; Asn 65→Asp;Ser 68→Gly; Leu 70→Ser; Arg 72→Gly; Lys 73→Ala; Asp 77→Val; Trp 79→Arg;Arg 81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr 100→Pro; Leu 103→Gly; Tyr106→Thr; Lys 125→His; Ser 127→Leu; Tyr 132→Phe; Lys 134→Glu; (k) Gln28→His; Leu 36→Gln; Ala 40→Tyr; Gln 49→Val; Tyr 52→Trp; Asn 65→Asp; Ser68→Glu; Leu 70→Ser; Arg 72→Gly; Lys 73→Pro; Asp 77→Asn; Trp 79→Arg; Arg81→Trp; Cys 87→Ser; Asn 96→Gly; Tyr 100→Leu; Leu 103→Gly; Tyr 106→Thr;Lys 125→His; Ser 127→Leu; Tyr 132→Phe; Lys 134→Glu; (l) Gln 28→His; Leu36→Val; Ala 40→Trp; Ile 41→Tyr; Gln 49→Ile; Tyr 52→Thr; Asn 65→Asp; Ser68→Gln; Leu 70→Gly; Arg 72→Glu; Lys 73→Gln; Lys 74→Glu; Asp 77→Lys; Trp79→Ser; Arg 81→His; Cys 87→Ser; Tyr 100→Trp; Leu 103→Asp; Tyr 106→Pro;Asn 116→Asp; Lys 125→Gly; Ser 127→Met; Asn 129→Asp; Tyr 132→Val; Lys134→Ala; (m) Gln 28→His; Leu 36→Val; Ala 40→Trp; Ile 41→Tyr; Gln 49→Ile;Tyr 52→Thr; Asn 65→Asp; Ser 68→Gln; Leu 70→Gly; Arg 72→Glu; Lys 73→Gln;Lys 74→Glu; Asp 77→Lys; Trp 79→Ser; Arg 81→His; Cys 87→Ser; Asn 96→Asp;Tyr 100→Trp; Leu 103→Asp; Tyr 106→Pro; Lys 125→Gly; Val 126→Met; Ser127→Met; Asn 129→Asp; Tyr 132→Val; Lys 134→Ala; or (n) Gln 28→His; Leu36→Met; Ala 40→Trp; Ile 41→Asp; Gln 49→Ile; Tyr 52→Thr; Asn 65→Asp; Ser68→Gln; Leu 70→Ala; Arg 72→Glu; Lys 73→Gln; Asp 77→Lys; Trp 79→Ser; Arg81→His; Cys 87→Ser; Asn 96→Gln; Tyr 100→Trp; Leu 103→Asp; Tyr 106→Pro;Lys 125→Gly; Ser 127→Met; Tyr 132→Val; Lys 134→Ala.
 68. The nucleic acidmolecule of claim 59, wherein the mutein comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-14.
 69. Thenucleic acid molecule of claim 59, wherein the mutein has at least 90%identity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-14.
 70. The nucleic acid molecule of claim 59, wherein themutein is capable of binding angiopoietin-1 (Ang-1) with detectableaffinity.
 71. The nucleic acid molecule of claim 70, wherein the muteinis capable of binding Ang-1 with an affinity measured by an IC50 valueof about 150 nM or lower.
 72. The nucleic acid molecule of claim 70,wherein the mutein comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-14.
 73. The nucleic acid molecule ofclaim 70, wherein the mutein has at least 90% identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-14.
 74. Thenucleic acid molecule of claim 59, wherein the mutein is cross-reactivewith both human Ang-2 and mouse Ang-2.
 75. The nucleic acid molecule ofclaim 74, wherein the mutein is capable of binding mouse Ang-2 with anaffinity measured by an IC50 value of about 5 nM or lower.
 76. Thenucleic acid molecule of claim 59, wherein the mutein is capable ofblocking the binding of human Ang-2 to human Tie-2 with an IC50 value ofabout 25 nM or lower.
 77. The nucleic acid molecule of claim 59, whereinthe mutein is fused at its N-terminus and/or its C-terminus, with orwithout an intervening linker, to a fusion partner.
 78. A cellcontaining the nucleic acid molecule of claim
 59. 79. A method ofinhibiting or reducing angiogenesis in a subject, comprisingadministering to said subject an effective amount of a mutein encoded bya nucleotide sequence of claim 59 or a composition comprising suchmutein.